Boysenberry, apple, and blackcurrant compositions and methods of preparation and use therefor

ABSTRACT

The present disclosure encompasses compositions prepared from Boysenberry and apple, as well as compositions prepared from Boysenberry, apple and blackcurrant. Also encompassed are methods of preparing these compositions and methods of using these compositions, in particular, for treating or preventing disorders of the respiratory system, including amongst others: inflammation, asthma, chronic obstructive pulmonary disease, allergic airways inflammation, reactive airway disease, airway fibrosis, and airway remodelling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage Application of InternationalPatent Application No. PCT/NZ2018/050109 filed Aug. 8, 2018 which claimsthe benefit of New Zealand Patent Application No. 734440 filed g Aug. 8,2017, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to compositions prepared from Boysenberryand apple, and compositions prepared from Boysenberry, apple andblackcurrant. The present disclosure relates also to methods ofpreparing such compositions, and methods of using such compositions,including methods of treating or preventing disorders of the respiratorytract, such as inflammatory conditions of the respiratory tract,including asthma, chronic obstructive pulmonary disease, allergicairways inflammation, reactive airway disease, airway fibrosis, andairway remodelling and the physiological conditions that lead to theseconditions.

BACKGROUND OF THE INVENTION

Airway remodelling is understood as a progressive and irreversibledecline in airway function due to chronic inflammatory processes thatresult in structural changes in the airway walls (67). Remodelling ofthe airways may involve all layers of the airway walls and can occuranywhere along the respiratory tract, from the large to the smallairways. Remodelling leads to key changes in epithelial tissue (68).Damaged epithelial cells release profibrotic cytokines, including EGFand TGF-β, which leads to fibroblast proliferation, myofibroblastactivation, and ultimately to the formation of subepithelial fibrosis(69). Airway smooth muscle hypertrophy and hyperplasia lead to anincrease in airway wall thickness. In turn, this leads to acceleratedlung function decline and irreversible or only partially reversibleairflow obstruction.

Acute disorders causing airway inflammation include asthma and COPD. Itis estimated that 150 million people are affected by asthma worldwide,with a 5-15% prevalence in children (61). The prevalence of COPD isestimated to be between 15-20%, and it is estimated to cause 2.75million deaths per annum (86). In the case of chronic asthma there isevidence of cumulative tissue remodelling, fibrosis, and consequent lossof lung function (45, 59). Fibrosis and remodelling are also associatedwith COPD. Remodelling manifests as a progressive increase in symptomssuch as dyspnoea and a corresponding decrease in bronchodilatorresponsiveness (67). Current asthma treatments are designed to manageinflammation and mitigate the symptoms and severity of asthma attacks(30, 43). COPD treatments are also designed to control inflammation andimprove airflow. However, no asthma or COPD medications are known toprevent airway remodelling (70-74), and there are no current treatmentsavailable to prevent aberrant remodelling.

Asthma pathogenesis and lung tissue remodelling have been linked to anincrease in profibrotic, arginase-positive, alternatively activatedmacrophages (AAMs) in the lung (27, 29, 34). However, temporal depletionof macrophage populations in a model of bleomycin-induced pulmonaryfibrosis illustrates that lung macrophages may also develop fibrolyticfunctions that contribute toward the resolution of fibrosis (14).

Mediators of tissue remodelling, such as the matrix metalloproteinases(MMPs), play an important role in regulating fibrosis (5, 7, 8, 10, 38).Of these, MMP-9 is widely reported to increase in conditions of lunginflammation and fibrosis and is associated with improved symptoms inasthma sufferers (25, 32, 33). MMP-9, in concert with other MMPs, exertsfibrolytic activity that leads to the breakdown of denatured collagensthat could moderate inappropriate lung remodelling (5, 60). As such,MMP-9 may represent a possible therapeutic target to limit lung damagein chronic asthma as well as other pulmonary diseases.

Large epidemiological studies have found that increased fruit andvegetable consumption correlates with reduced asthma symptoms (39, 46,47). These population studies have identified foods high in polyphenolssuch as apples, pears (13, 51, 62), carrots, tomatoes (46-48), andcitrus (11) as having inverse correlations with frequency and severityof reported asthma symptoms, in particular wheeze and cough symptoms(11, 13, 46, 47). However, the effect of fruits high in polyphenols onlung fibrosis and tissue remodelling is unknown. To date, no generallysuccessful methods for preventing airway remodelling have beenestablished.

Given the occurrence of respiratory disorders in the population,including allergic airways inflammation associated with asthma, COPD,reactive airway disease, airway fibrosis, and airway remodelling, thereis a need for new compositions, particularly compositions derived fromnatural sources, for restoring and maintaining respiratory health.

SUMMARY OF THE INVENTION

In one aspect, the invention encompasses a method of treating orpreventing inflammation in the respiratory tract, comprising:administering to a subject a composition comprising a Boysenberry andapple concentrate or a Boysenberry, apple and blackcurrant concentrate,thereby treating or preventing the inflammation in the respiratory tractin the subject.

Also encompassed is a composition, for example, a nutraceuticalcomposition, comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for treating orpreventing inflammation in the respiratory tract in a subject.

In one other aspect, the invention encompasses a method of treating orpreventing asthma, comprising: administering to a subject a compositioncomprising a Boysenberry and apple concentrate or a Boysenberry, appleand blackcurrant concentrate, thereby treating or preventing the asthmain the subject.

Also encompassed is a composition, for example, a nutraceuticalcomposition, comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for treating orpreventing asthma in a subject.

In yet one other aspect, the invention encompasses a method of treatingor preventing chronic obstructive pulmonary disease, comprising:administering to a subject a composition comprising a Boysenberry andapple concentrate or a Boysenberry, apple and blackcurrant concentrate,thereby treating or preventing the chronic obstructive pulmonary diseasein the subject.

Also encompassed is a composition, for example, a nutraceuticalcomposition, comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for treating orpreventing chronic obstructive pulmonary disease in a subject.

In still one other aspect, the invention encompasses a method oftreating or preventing aberrant collagen deposition or fibrosis in therespiratory tract, comprising: administering to a subject a compositioncomprising a Boysenberry and apple concentrate or a Boysenberry, appleand blackcurrant concentrate, thereby treating or preventing theaberrant collagen deposition or fibrosis in the respiratory tract of thesubject.

Also encompassed is a composition, for example, a nutraceuticalcomposition, comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for treating orpreventing aberrant collagen deposition or fibrosis in a subject.

In even one other aspect, the invention encompasses a method of treatingor preventing airway remodelling, comprising: administering to a subjecta composition comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate, thereby treating orpreventing the airway remodelling in the subject.

Also encompassed is a composition, for example, a nutraceuticalcomposition, comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for treating orpreventing airway remodelling in a subject.

In various aspects:

The composition comprises Boysenberry juice concentrate, Boysenberrypuree, or Boysenberry powder.

The composition comprises apple juice concentrate, apple puree, or applepowder.

The composition comprises blackcurrant juice concentrate, blackcurrantpuree, or blackcurrant powder.

The composition comprises a dosage unit comprising about 5 to about 500mg total anthocyanins.

For the nutraceutical composition comprising the Boysenberry and appleconcentrate, the composition comprises a dosage unit comprising about 5to about 500 mg total Boysenberry anthocyanins.

For the nutraceutical composition comprising the Boysenberry, apple andblackcurrant concentrate, the composition comprises a dosage unitcomprising about 5 to about 500 mg total Boysenberry and blackcurrantanthocyanins.

The composition is formulated for enteral administration.

The composition is formulated for oral administration.

The composition is formulated as a syrup or as drops.

The composition is formulated as a gel or jelly.

The composition is formulated as a tablet or capsule.

The composition is formulated for administration at a dosage of about0.1 mg/kg to about 10 mg/kg total anthocyanins/subject's body weight.

For the nutraceutical composition comprising the Boysenberry and appleconcentrate, the composition is formulated for administration at adosage of about 0.1 mg/kg to about 10 mg/kg total Boysenberryanthocyanins/subject's body weight.

For the nutraceutical composition comprising the Boysenberry, apple andblackcurrant concentrate, the composition is formulated foradministration at a dosage of about 0.1 mg/kg to about 10 mg/kg totalBoysenberry and blackcurrant anthocyanins/subject's body weight.

The composition is formulated for administration at a dosage of about 10mg to about 1000 mg total anthocyanins per day.

For the nutraceutical composition comprising the Boysenberry and appleconcentrate, the composition is formulated for administration at adosage or about 10 mg to about 1000 mg total Boysenberry anthocyaninsper day.

For the nutraceutical composition comprising the Boysenberry, apple andblackcurrant concentrate, the composition is formulated foradministration at a dosage of or about 10 mg to about 1000 mg totalBoysenberry and blackcurrant anthocyanins per day.

Alternatively, the dosage is about 10 mg to about 200 mg totalanthocyanins per day, or about 50 mg total anthocyanins per day.

For the nutraceutical composition comprising the Boysenberry and appleconcentrate, the dosage is about 10 mg to about 200 mg total Boysenberryanthocyanins per day, or about 50 mg total Boysenberry anthocyanins perday.

For the nutraceutical composition comprising the Boysenberry, apple andblackcurrant concentrate, the dosage is about 10 mg to about 200 mgtotal Boysenberry and blackcurrant anthocyanins per day, or about 50 mgtotal Boysenberry and blackcurrant anthocyanins per day.

The composition comprises added polyphenols.

The composition is formulated for co-administration with a furtherrespiratory aid.

The composition is formulated for co-administration with one or moretreatments for a chronic respiratory disorder.

The inflammation is associated with a chronic respiratory disorder.

The inflammation is associated with one or more of: asthma, chronicobstructive pulmonary disease, allergic airways inflammation, reactiveairway disease, airway fibrosis, and airway remodelling.

The asthma is atopic or non-atopic.

The asthma is associated with airway fibrosis or airway remodelling.

The chronic obstructive pulmonary disease is associated with smoking orpollution.

The chronic obstructive pulmonary disease is associated with airwayfibrosis or airway remodelling.

The aberrant collagen deposition or the fibrosis is associated with achronic respiratory disorder.

The aberrant collagen deposition or the fibrosis is associated withasthma or chronic obstructive pulmonary disease.

The airway remodelling is associated with a chronic respiratorydisorder.

The airway remodelling is associated with one or more of: asthma andchronic obstructive pulmonary disease.

In still one further aspect, the invention comprises the use of acomposition comprising a Boysenberry and apple concentrate or aBoysenberry, apple and blackcurrant concentrate for preparing anutraceutical composition for:

(i) treating or preventing inflammation in a respiratory tract in asubject;

(ii) treating or preventing asthma in a subject;

(iii) treating or preventing chronic obstructive pulmonary disease in asubject;

(iv) treating or preventing allergic airways inflammation in a subject;

(v) treating or preventing reactive airway disease in a subject;

(vi) treating or preventing aberrant collagen deposition in a subject;

(vii) treating or preventing fibrosis in a respiratory tract in asubject;

(viii) treating or preventing airway remodelling in a subject.

In various aspects, the therapeutic use employs the compositions,dosages, and formulations, and relates to the various conditions, asnoted above.

The foregoing brief summary broadly describes the features and technicaladvantages of certain embodiments of the present invention. Furthertechnical advantages will be described in the detailed description ofthe invention and examples that follows.

Novel features that are believed to be characteristic of the inventionwill be better understood from the detailed description of the inventionwhen considered in connection with any accompanying figures andexamples. However, the figures and examples provided herein are intendedto help illustrate the invention or assist with developing anunderstanding of the invention, and are not intended to limit theinvention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D. Therapeutic oral Boysenberry treatment reduces OVA-inducedchronic lung inflammation. A: 6-week-old male C57Bl/6 mice (n=10 pergroup) were primed i.p. with OVA/alum (day 0) then challenged i.n. withOVA every 7 days for 10 weeks. From weeks 6 to 10 Boysenberry juice wasadministered orally (gavage) 1 h prior to, and 2 days after, each i.n.OVA challenge. B: representative H&E staining of lung tissue from naive,10-week OVA challenge only (OVA), 10-week OVA challenge with therapeuticBoysenberry (OVA BoysB) treatment, and Boysenberry alone (BoysB)-treatedmice. Arrows and * indicate immune cell infiltrate. Magnification ×4(top) and ×10 (bottom). C: representative AB-PAS staining of lungtissue. Arrows indicate dark purple mucus-positive bronchioles.Magnification ×4 (top) and ×20 (bottom). D: total cells per ml BALF andflow cytometric quantification of percentage of eosinophils in BALFfollowing final OVA challenge. **P<0.01, ***P<0.001 (n=10 per group)one-way ANOVA with Tukey's post hoc test compared with naive and OVAchallenge with therapeutic Boysenberry treatment and Boysenberryalone-treated mice.

FIG. 2A-D. Boysenberry treatment increases arginase expression andmacrophage accumulation in lung tissue during OVA-induced chronic lunginflammation. A: representative H&E staining of lung tissue from 10-weekOVA-challenged mice, with and without Boysenberry treatment. Arrowsindicate macrophages. Magnification ×100, scale 200 μm. B:representative Western blot analysis of iNOS (135 kDa) and arginase (37kDa) expression in lung tissue. Noncontiguous bands from the sameWestern blot are shown. C and D: quantification of iNOS and arginaseWestern blot signals normalized to β-actin signal. **P<0.01 (n=10 pergroup) one-way ANOVA with Tukey's post hoc test.

FIG. 3A-B. Boysenberry treatment increases the accumulation ofarginase+alternatively activated macrophages during OVA-induced chroniclung inflammation. Representative immunofluorescent labelling of lungtissue from 10-week OVA-challenged mice with and with-out Boysenberrytreatment. A: CD68+CD206+macrophages identified by *. B:CD206+arginase+macrophages identified by *. DAPI nuclear stain (darkblue). Magnification ×40, scale 200 μm.

FIG. 4A-E. Boysenberry treatment decreases collagen deposition andincreases MMP-9 protein expression in lung tissue during OVA-inducedchronic lung inflammation. A: representative Masson's trichromestaining. Magnification ×40, scale 200 B: hydroxyproline levels (mg/glung tissue); ***P<0.001 (n=10) one-way ANOVA with Tukey's post hoctest. C: lung TGFβ concentration as determined by ELISA; *P 0.05 (n 10per group) one-way ANOVA with Tukey's post hoc test. D: Western blotanalysis of MMP-9 (pro 105 kDa; active 92 kDa) and TIMP-1 (29 kDa)expression (noncontiguous bands from the same Western blot are shown) inlung tissue from 10-week OVA-challenged mice with and withoutBoysenberry treatment. E: ratio of TIMP-1/MMP-9 protein expressionnormalized to β-actin loading control; **P<0.01 (n=10) one-way ANOVAwith Tukey's post hoc test compared with naive and OVA plus Boysenberrytreatment.

FIG. 5A-B. Boysenberry treatment increases MMP-9 expression byalternatively activated macrophages in lung tissue during OVA-inducedchronic lung inflammation. A: DAB labelling of MMP-9+macrophages(arrows). B: immunofluorescent labelling of CD206+MMP-9+macrophages (*).DAPI nuclear stain (dark blue). Magnification ×40, scale 200 μm.

FIG. 6A-B. Depletion of lung macrophages reduced the effect of oralBoysenberry treatment on OVA-induced chronic lung inflammation. A:6-week-old male C57Bl/6 mice (n=10 per group) were primed i.p. withOVA/alum (day 0) then challenged i.n. with OVA every 7 days for 5 weeks.From weeks 6 to 7 macrophages were depleted using clodronate liposomes(CloLip) the day before Boysenberry juice was administered orally(gavage). B: flow cytometric quantification of percentage of macrophagesin BALF following final clodronate macrophage depletion; *P<0.05 (n=10per group) one-way ANOVA with Tukey's post hoc test. C: hydroxyprolinelevels (mg/g lung tissue) in the lung; *P<0.05 (n=10 per group) one-wayANOVA with Tukey's post hoc test.

FIG. 7A-G. Prophylactic oral Boysenberry treatment reduces OVA-inducedchronic lung inflammation and collagen deposition. A: 6-week-old maleC57Bl/6 mice (n=10 per group) were primed i.p. with OVA/alum thenchallenged i.n. with OVA every 7 days for 5 weeks. Boysenberry juice wasadministered orally (gavage) 1 h prior and 2 days after each i.n. OVAchallenge. B: lung tissue was stained with total cells per ml BALF andflow cytometric quantification of percentage of eosinophils in BALFfollowing final OVA challenge; *P<0.05, **P<0.01 (n=10 per group)one-way ANOVA with Tukey's post hoc test. C: AB-PAS, dark purplemucus-positive bronchioles (arrows); magnification ×20, scale 200 μm. D:Masson's trichrome; magnification ×40, scale 200 μm. E: hydroxyprolinelevels (mg/g lung tissue) in the lung. *P<0.05, **P<0.01 (n=10 pergroup) one-way ANOVA with Tukey's post hoc test. F: Western blotanalysis of iNOS, arginase, MMP-9, and TIMP-1 lung tissue. Noncontiguousbands from the same Western blot are shown. G: ratio of TIMP-1/MMP-9protein levels normalized to β-actin loading control. *P<0.05, (n=10 pergroup) one-way ANOVA with Tukey's post hoc test. FIG. 4 : Schematic oftrial treatments, washouts, and sampling points.

FIG. 8 . Schematics for experiments testing Boysenberry treatmentcombinations in a model of acute allergic airways inflammation.

FIG. 9 . Treatment utilising BerriQi™ Boysenberry with appleadministration reduced immune cell numbers in model of acute allergicairways inflammation. Total cell infiltration into the lung followingovalbumin (OVA)-induced allergic airways infiltration. Totalbronchioalveolar lavage fluid (BALF) cell numbers were determined 4 dayspost-OVA challenge (n=10 per intervention group).

FIG. 10 . Treatment utilising BerriQi™ Boysenberry with appleadministration reduced eosinophil numbers in model of acute allergicairways inflammation. Eosinophil infiltration into the lung following anovalbumin (OVA)-induced allergic airways infiltration. Totalbronchioalveolar lavage fluid (BALF) eosinophil cell numbers weredetermined 4 days post-OVA challenge (n=10 per intervention group).BerriQi™ concentrates and the other concentrates are described inExample 2, below.

FIG. 11 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation.Haematoxylin and eosin staining of lung tissues following appletreatment.

FIG. 12 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation.Haematoxylin and eosin staining of lung tissues following Boysenberrytreatment.

FIG. 13 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation.Haematoxylin and eosin staining of lung tissues following BerriQi™Boysenberry with apple treatment. Representative 10× (A-D) and 20× (E-H)images of (A, E) naïve, (B, F) OVA, (C, G) BerriQi™ Boysenberry withapple 10, and (D, H) BerriQi™ Boysenberry with apple 1. Immune cellsappear as dark pink/purple clusters.

FIG. 14 . Treatment utilising BerriQi™ Boysenberry with appleadministration reduced neutrophil numbers in model of acute allergicairways inflammation. Neutrophil infiltration into the lung followingovalbumin (OVA)-induced allergic airways infiltration. Totalbronchioalveolar lavage fluid (BALF) neutrophil cell numbers weredetermined 4 days post-OVA challenge (n=10 per intervention group).

FIG. 15 . Treatment utilising BerriQi™ Boysenberry with appleadministration reduced monocyte numbers in model of acute allergicairways inflammation. Monocyte infiltration into the lung following anovalbumin (OVA)-induced allergic airways infiltration. Totalbronchioalveolar lavage fluid (BALF) monocyte cell numbers weredetermined 4 days post-OVA challenge (n=10 per intervention group).

FIG. 16 . Treatment utilising BerriQi™ Boysenberry with appleadministration reduced antigen presenting cells in model of acuteallergic airways inflammation. Antigen Presenting Cell (APC)infiltration into the lung following an ovalbumin (OVA)-induced allergicairways infiltration. Total bronchioalveolar lavage fluid (BALF) APCnumbers were determined 4 days post-OVA challenge (n=10 per interventiongroup).

FIG. 17 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Alcianblue/periodic acid Schiff diastase staining of lung tissues followingapple treatment.

FIG. 18 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Alcianblue/periodic acid Schiff diastase staining of lung tissues followingBoysenberry treatment.

FIG. 19 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Alcianblue/periodic acid Schiff diastase staining of lung tissues followingBerriQi™ Boysenberry with apple treatment. Representative 10× (A-D) and20× (E-H) images of images of (A, E) naïve, (B, F) OVA, (C, G) BerriQi™Boysenberry with apple 10, and (D, H) BerriQi™ Boysenberry with apple 1.Mucous positive goblet cells are dark purple.

FIG. 20 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Masson'strichrome staining of lung tissues following apple treatment.

FIG. 21 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Masson'strichrome staining of lung tissues following Boysenberry treatment.

FIG. 22 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. Masson'strichrome staining of lung tissues following BerriQi™ Boysenberry withapple treatment. Representative 10× (A-D) and 20× (E-H) images of imagesof (A, E) naïve, (B, F) OVA, (C, G) BerriQi™ Boysenberry with apple 10,and (D, H) BerriQi™ Boysenberry with apple 1.

FIG. 23 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation.Granulocyte-macrophage colony-stimulating factor levels followingBoysenberry, apple, and BerriQi™ Boysenberry with apple treatments.

FIG. 24 . Treatment utilising BerriQi™ Boysenberry with appleadministration in model of acute allergic airways inflammation. CCL11levels following Boysenberry, apple, and BerriQi™ Boysenberry with appletreatments.

FIG. 25 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration reduced immune cell numbers in model of chronic allergicairways inflammation. Total cell infiltration into the lung followingchronic ovalbumin (OVA)-induced allergic airways infiltration. Totalbronchioalveolar lavage fluid (BALF) cell numbers were determined 4 daysfollowing final OVA challenge. Data are mean±SEM (n=20 per interventiongroup). P<0.05, P<0.001 compared to OVA; P<0.01 compared to naïve.

FIG. 26 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration reduced eosinophil numbers in model of chronic allergicairways inflammation. Eosinophil infiltration into the lung followingchronic ovalbumin (OVA)-induced allergic airways infiltration. Number ofeosinophils in bronchioalveolar lavage fluid (BALF) cell was determined4 days following final OVA challenge. Data are mean±SEM (n=20 perintervention group). P<0.001 compared to naïve.

FIG. 27 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration reduced antigen presenting cell numbers in model ofchronic allergic airways inflammation. Antigen presenting cell (APC)infiltration into the lung following chronic ovalbumin (OVA)-inducedallergic airways infiltration. Number of APCs in bronchioalveolar lavagefluid (BALF) cell was determined 4 days following final OVA challenge.Data are mean±SEM (n=20 per intervention group). P<0.01 compared tonaïve.

FIG. 28 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration reduced monocyte numbers in model of chronic allergicairways inflammation. Monocyte infiltration into the lung followingchronic ovalbumin (OVA)-induced allergic airways infiltration. Number ofmonocytes in bronchioalveolar lavage fluid (BALF) cell was determined 4days following final OVA challenge. Data are mean±SEM (n=20 perintervention group).

FIG. 29 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration in model of chronic allergic airways inflammation.Photomicrographs of lung tissue sections stained with haematoxylin andeosin staining following chronic ovalbumin (OVA)-induced allergicairways infiltration. Mice were primed with OVA/Alum intraperitoneallyand then challenged 7 days later with OVA intranasally for 10 weeks.After 5 weeks mice were orally gavaged with nothing (A—naïve) water(B—OVA control) 100% (C) 50% (D) or 25% (E) BerriQi™ Boysenberry withapple 2 days prior, 1 hour before an OVA challenge and again 2 dayspost-challenge for 5 weeks.

FIG. 30 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration in model of chronic allergic airways inflammation.Photomicrographs of lung tissue sections stained with Alcian blue andperiodic acid-Schiff staining of lung tissue following chronic ovalbumin(OVA)-induced allergic airways infiltration. Mice were primed withOVA/Alum intraperitoneally and then challenged 7 days later with OVAintranasally for 10 weeks. After 5 weeks mice were orally gavaged withnothing (A—naïve) water (B—OVA control) 100% (C) 50% (D) or 25% (E)BerriQi™ Boysenberry with apple 2 days prior, 1 hour before an OVAchallenge and again 2 days post-challenge for 5 weeks.

FIG. 31 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration in model of chronic allergic airways inflammation.Photomicrographs of lung tissue sections stained with Masson's Trichromestaining of lung tissue following chronic ovalbumin (OVA)-inducedallergic airways infiltration. Mice were primed with OVA/Alumintraperitoneally and then challenged 7 days later with OVA intranasallyfor 10 weeks. After 5 weeks mice were orally gavaged with nothing(A—naïve) water (B—OVA control) 100% (C) 50% (D) or 25% (E) BerriQi™Boysenberry with apple 2 days prior, 1 hour before an OVA challenge andagain 2 days post-challenge for 5 weeks.

FIG. 32 . Treatment utilising BerriQi™ Boysenberry with apple (BA)administration in model of chronic allergic airways inflammation.Quantification of collagen in the lung following chronic ovalbumin(OVA)-induced allergic airways infiltration. Mice were primed withOVA/Alum intraperitoneally and then challenged 7 days later with OVAintranasally for 10 weeks. After 5 weeks mice were orally gavaged withnothing (naïve), water (OVA control), 100%, 50%, or 25% BerriQi™Boysenberry with apple (BA) 2 days prior, 1 hour before an OVA challengeand again 2 days post-challenge for 5 weeks. Collagen was determinedusing the hydroxyproline assay 4 days following the final OVA challenge.Data are mean±SEM (n=20 per intervention group). P<0.001 compared to allother treatment groups.

FIG. 33 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration reduced immune cell numbers in model ofchronic allergic airways inflammation. Total cell infiltration into thelung following chronic ovalbumin (OVA)-induced allergic airwaysinfiltration. Total bronchioalveolar lavage fluid (BALF) cell numberswere determined 4 days following final OVA challenge (n=20 perintervention group).

FIG. 34 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration reduced eosinophil numbers in model ofchronic allergic airways inflammation. Eosinophil infiltration into thelung following chronic ovalbumin (OVA)-induced allergic airwaysinfiltration. Number of eosinophils in bronchioalveolar lavage fluid(BALF) cell was determined 4 days following final OVA challenge (n=20per intervention group). P<0.001 compared to naïve.

FIG. 35 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration reduced antigen presenting cell numbers inmodel of chronic allergic airways inflammation. Antigen presenting cell(APC) infiltration into the lung following chronic ovalbumin(OVA)-induced allergic airways infiltration. Number of APCs inbronchioalveolar lavage fluid (BALF) cell was determined 4 daysfollowing final OVA challenge (n=20 per intervention group). P<0.001,P<0.01 compared to OVA.

FIG. 36 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration reduced monocyte numbers in model of chronicallergic airways inflammation. Monocyte infiltration into the lungfollowing chronic ovalbumin (OVA)-induced allergic airways infiltration.Number of monocytes in bronchioalveolar lavage fluid (BALF) cell wasdetermined 4 days following final OVA challenge (n=20 per interventiongroup).

FIG. 37 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration in model of chronic allergic airwaysinflammation. Haematoxylin and eosin staining of lung tissue followingovalbumin (OVA)-induced allergic airways infiltration. Mice were primedwith OVA/Alum intraperitoneally and then challenged 7 days later withOVA intranasally for 10 weeks. After 5 weeks mice were orally gavagedwith nothing (naïve) water (OVA control) or 100% BerriQi™ Boysenberrywith apple and blackcurrant (OVA+100%) 2 days prior, 1 hour before anOVA challenge and again 2 days post-challenge for 5 weeks.

FIG. 38 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration in model of chronic allergic airwaysinflammation. Alcian blue and periodic acid-Schiff staining of lungtissue following ovalbumin (OVA)-induced allergic airways infiltration.Mice were primed with OVA/Alum intraperitoneally and then challenged 7days later with OVA intranasally for 10 weeks. After 5 weeks mice wereorally gavaged with nothing (naïve) water (OVA control) or 100% BerriQi™Boysenberry with apple and blackcurrant (OVA+100%) 2 days prior, 1 hourbefore an OVA challenge and again 2 days post-challenge for 5 weeks.

FIG. 39 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration in model of chronic allergic airwaysinflammation. Masson's trichrome staining of lung tissue followingovalbumin (OVA)-induced allergic airways infiltration. Mice were primedwith OVA/Alum intraperitoneally and then challenged 7 days later withOVA intranasally for 10 weeks. After 5 weeks mice were orally gavagedwith nothing (naïve) water (OVA control) or 100% BerriQi™ Boysenberrywith apple and blackcurrant (OVA+100%) 2 days prior, 1 hour before anOVA challenge and again 2 days post-challenge for 5 weeks.

FIG. 40 . Treatment utilising BerriQi™ Boysenberry with blackcurrant andapple (BBA) administration in model of chronic allergic airwaysinflammation. Quantification of collagen in the lung following ovalbumin(OVA)-induced allergic airways infiltration. Mice were primed withOVA/Alum intraperitoneally and then challenged 7 days later with OVAintranasally for 10 weeks. After 5 weeks mice were orally gavaged with100% BerriQi™ Boysenberry with apple and blackcurrant (100% BBA) 2 daysprior, 1 h before an OVA challenge and again 2 days post-challenge for 5weeks. Collagen was determined using the hydroxyproline assay wasdetermined 4 days post-OVA challenge (n=20 per intervention group).

FIG. 41 is the dosing/challenge regimen for Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The following description sets forth numerous exemplary configurations,parameters, and the like. It should be recognised, however, that suchdescription is not intended as a limitation on the scope of the presentinvention, but is instead provided as a description of exemplaryembodiments.

All references, including patents and patent applications, cited in thisspecification are hereby incorporated by reference. No admission is madethat any reference constitutes prior art. Nor does discussion of anyreference constitute an admission that such reference forms part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

Definitions

In each instance herein, in descriptions, embodiments, and examples ofthe present invention, the terms “comprising”, “including”, etc., are tobe read expansively, without limitation. Thus, unless the contextclearly requires otherwise, throughout the description and the claims,the words “comprise”, “comprising”, and the like are to be construed inan inclusive sense as to opposed to an exclusive sense, that is to sayin the sense of “including but not limited to”.

The term “consisting essentially of”, as used herein, may refer to thepresence of a concentrate in a composition. For example, the concentratemay be at least 80% by weight of the composition, or at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.8%, or at least 99.9% by weight of the composition (% w/w). Forliquids, the concentrate may be at least 80% by volume of thecomposition volume, or at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.8%, or at least99.9% by volume of the composition volume (% v/v).

In the present description, the articles “a” and “an” are used to referto one or to more than one (i.e., to at least one) of the grammaticalobject of the article. By way of example, “an element” can be taken tomean one element or more than one element.

Throughout this description, the term “about” is used to indicate that avalue includes the standard deviation of error for the method beingemployed to determine the value, for example, levels of compounds ordosage levels, as described in detail herein. In particular, the term“about” encompasses a 10% to 15% deviation (positive and negative) inthe stated value or range, particularly 10% deviation (positive andnegative) in the stated value or range.

“Airway remodelling”, also referred to as tissue or lung remodelling,refers to the development of specific structural changes in the airwaywall. This may include, for example, remodelling of the fibrousconnective tissue in the lining of the airways, for example, in thelungs. Airway remodelling may include one or more of subepithelialfibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia,and hypertrophy, mucous gland and goblet cell hyperplasia, andepithelial disruption. Symptoms may include decreased airwaydistensibility (i.e., stiffer airways), diminished elastic recoil,progressive decline in FEV1 (forced expiratory volume 1), and FVC(forced vital capacity), accelerated lung function decline, irreversibleor only partially reversible airflow obstruction, dyspnoea, anddecreased responsiveness to respiratory therapy (e.g., asthma or COPDtherapeutics).

“Asthma” refers to an inflammatory disorder of the airways of the lungs,characterized by variable and recurring breathing impairment, includingairflow obstruction and bronchospasm. Airflow obstruction may be definedas reduced FEV1 and/or reduced FEV1/VC ratio. The airflow obstruction inasthma may be reversible with or without medication. Symptoms of asthmamay include one or more of wheezing, coughing, chest tightness or pain,and shortness of breath. Included herein are atopic (e.g., allergen orantigen induced) and non-atopic forms of asthma, as well asexercise-induced asthma, occupational asthma, aspirin-induced asthma,and alcohol-induced asthma.

A “respiratory aid” is a composition that assists with airway functionor other aspects of the respiratory system, e.g., medicines, herbalcompositions, essential oils, and various compositions for inhalation.

“Airway”, “respiratory tract”, and “respiratory system” refer to any ofthe organs, tissues, or cellular components involved in gas exchange(i.e., breathing). This includes the upper respiratory tract, trachea,bronchi, bronchioles, alveoli, lungs, pleura and pleural cavity, and thenerves and muscles of breathing. It will be understood that “airways”describes the various structural components (e.g., cellular components,tissues, and organs) as well as the space where gas exchange occurs.

“Apple” as used herein encompasses any fruit of the genus Malus, and anyhybrid, variety, and genetic derivative thereof. Included, specifically,are Malus pumila, as well as the particular cultivars of ‘Gala’ ‘GoldenSupreme’ ‘McIntosh’ ‘Transparent’ ‘Primate’ ‘Sweet Bough’ ‘Duchess’‘Fuji’ ‘Jonagold’ ‘Golden Delicious’ ‘Red Delicious’ ‘Chenango’‘Gravenstein’ ‘McIntosh’ ‘Snow’ ‘Blenheim’ ‘Winesap’ ‘Granny Smith’‘King’ ‘Wagener’ ‘Swayzie’ ‘Greening’ and ‘Tolman Sweet’. Included alsoare the cultivars ‘Alice’ ‘Ambrosia’ ‘Ananasrenette’ ‘Aroma’ ‘Discovery’‘Envy’ ‘Braeburn’ ‘Bramley’ ‘Arkansas Black’ ‘Dougherty’/‘Red Dougherty’‘Goldrenette’/‘Reinette’ ‘Jazz’ ‘Jonagold’ ‘James Grieve’ ‘YellowTransparent’ ‘Pacific rose’ ‘Lobo’ ‘Sampion’/‘Shampion’ ‘Sonya’‘Splendour’/‘Splendor’ ‘Summerred’ ‘Pink Lady’ ‘Belle de Boskoop’ ‘CoxPomona’ ‘Cox's Orange Pippin’ ‘Kidd's Orange Red’ and ‘SugarBee’.Further included are the cultivars of ‘Haralson’ ‘Wealthy’ ‘Honeygold’and ‘Honeycrisp’. Included as well are crabapples, apple-crabapplehybrids, and cooking apples.

“Blackcurrant” as used herein encompasses any black/dark coloured Ribesfruit from the family Grossulariaceae, which includes but is not limitedto that of Ribes nigrum, Ribes nigrum L., Ribes americanum, Ribeshudsonianum, Ribes laxiforum, and Ribes×nidigrolaria. Any hybrid,variety, and genetic derivative of these are also included. Includedamongst suitable blackcurrant cultivars are those of ‘Andega’, ‘BenArd’, ‘Ben Alder’, ‘Ben Dorain’, ‘Ben Gairn’, ‘Ben Hope’, ‘Ben Lomand’,‘Ben More’, ‘Ben Rua’, ‘Ben Sarek’, ‘Ben Tirran’, ‘Blackadder’, ‘BlackDown’, ‘Burga’, ‘Ores’, ‘Magnus’, ‘Murchison’, ‘Sefton’, ‘Wellington’,‘Invigo’, ‘Titania’, ‘Consort’, ‘Crusader’, ‘Geant de Boskoop’, ‘Noir deBourgogne’, ‘Royal de Naples’, ‘Boskoop Giant’, ‘Tenah’, ‘Tiben’,‘Tines’, ‘Tisel’, and ‘Willoughby’.

“Boysenberry” as used herein encompasses a Rubus hybrid berry, whichincludes but is not limited to a berry obtained from the plantidentified as Rubus ursinus var loganobaccus cv Boysenberry, Rubusursinus×Rubus idaeus, Rubus loganbaccus×baileyanus Britt, and Rubusidaeus×Rubus ulmifolius. Generally speaking, a Boysenberry may bederived from a cross between raspberry and blackberry plants, or betweenraspberry, blackberry, and loganberry plants. Included are variousBoysenberry hybrids, varieties, and genetic derivatives thereof.Boysenberries are referred to herein as berries or, more broadly, asfruits.

“Chronic obstructive pulmonary disease”, or COPD, refers to a lungdisorder associated with progressive obstruction of the airways and poorairflow. Airflow obstruction may be defined as a reduction in FEV1and/or a reduction in FEV1/VC ratio. The airflow obstruction in chronicobstructive pulmonary disease may not be fully reversible. Symptomsinclude but are not limited to shortness of breath, cough, and sputumproduction (i.e., phlegm). COPD may be associated with smoking, airpollution, poorly ventilated cooking or heating fires. A geneticcomponent may also be involved in COPD. The disorder is also known aschronic obstructive lung disease (COLD), chronic obstructive airwaydisease (COAD), chronic bronchitis, pulmonary emphysema, amongst otherknown terminology.

“Concentrate”, for example, in relation to a Boysenberry, apple, orblackcurrant concentrate, or any combination thereof, refers to acomposition where the liquid component (e.g., juice) has been partly orsubstantially removed. Removal of a liquid component may be byevaporation or any other means. A concentrate may be prepared, forexample, as a puree, paste, or powder, or may be prepared from aBoysenberry, apple, or blackcurrant juice, or any combination thereof,e.g., prepared as a juice concentrate.

A “disorder” of respiratory tract includes a disease or other conditionaffecting any of the organs, tissues, or cellular components involved ingas exchange (i.e., breathing), as noted herein. The disorders may be anacute or chronic condition, such as inflammation and conditions that areassociated with inflammation. Particular disorders of interest includeasthma, chronic obstructive pulmonary disease, reactive airway disease,airway fibrosis, and airway remodelling. Other disorders are describedin detail herein.

A “genetic derivative” of a plant refers to offspring, sports, or othercultivars that are obtained from the parent stock. This includesoffspring obtained from a genetic cross with the parent, e.g., F1progeny or F2 progeny. The term “genetic derivative” may refer to thederived plant, itself, or to its fruit.

“Fibrosis”, as in airway or pulmonary fibrosis, refers to a disruptionin the regulation of collagen and other extracellular matrix componentsin the respiratory tract. In the airways of patients with fibrosis,there may be increased extracellular matrix deposition, such as in thereticular basement membrane region, lamina propria, and/or submucosa.Scar formation and the accumulation of excess fibrous connective tissueleads to thickening of the airway walls. Symptoms may include reducedoxygen supply, shortness of breath, chronic cough, fatigue and/orweakness, chest discomfort including chest pain, loss of appetite, andweight loss. Included are idiopathic forms of airway fibrosis, as wellas airway fibrosis associated with smoking, air pollution, connectivetissue disease (e.g., rheumatoid arthritis, sarcoidosis, etc),infections, medications (e.g., methotrexate, bleomycin, etc), andradiation therapy.

“Inflammation” refers to a condition characterised by one or more of:vasodilation, heat, redness, discomfort, swelling, edema, lesions,fissures, ulcerations, leukocyte extravasation, and loss of function.Included are both acute and chronic forms of inflammation, such as acuteairways inflammation, and other inflammatory disorders, e.g., autoimmunediseases or allergic conditions. Particularly included are asthma,chronic obstructive pulmonary disease, airway fibrosis, reactive airwaydisease, and airway remodeling. Other inflammatory disorders aredescribed elsewhere in this document.

As noted herein, the terms “lyophilising” and “freeze drying” are usedsynonymously. It will be understood that the terms “freezedrying”/“lyophilising” do not exclude the use of higher temperatures(i.e., higher than freezing temperatures). For example, highertemperatures may be used for removing residual moisture during thesecondary drying phase for lyophilisation/freeze drying procedures.

A “nutraceutical” refers to a standardised composition foradministration to a subject. It may be a pharmaceutical gradecomposition, and may maintain or improve the health of a subject, or maytreat or prevent one or more disorder in a subject.

“Reactive airway disease” refers to an inflammatory airway disordercharacterised by reversible airway narrowing due to external stimuli.The term can encompass other known disorders such as asthma, chronicobstructive pulmonary disease, upper respiratory tract infections, etc,or can refer to conditions that are similar to these disorders but notdirectly diagnosed as such, e.g., having asthma-like syndrome orasthma-like symptoms. Subjects with reactive airway disease may show oneor more symptoms of coughing, wheezing, or shortness of breath uponexposure to particular stimuli, for example, smoke, vapour, fume, orother irritants.

As used herein, a “subject” may be a human or non-human animal,particularly a mammal, including cattle, sheep, goats, pigs, horses, andother livestock, including, as well, dogs, cats, and other domesticatedpets. In particular aspects, the subject is a human being.

“Treating” as used herein is meant as reducing, ameliorating, orresolving a disorder, for example a respiratory disorder, such as adisease or other condition of the respiratory system. A treatment willresult in the reduction, amelioration, or elimination of one or moresymptoms of the disorder.

“Preventing” as used herein is meant as stopping or delaying the onsetof a disorder, for example a respiratory disorder, such as a disease orother condition of the respiratory system. A preventative measure willresult in the stoppage or delay of one or more symptoms of the disorder,or a lessening of symptoms if such do arise. It should be understoodthat the term “treating or preventing” does not exclude the possibilityof obtaining both treatment and prevention (e.g., at the same time or atdifferent times) of a disorder in any given subject. In the same waytreatment of “asthma or fibrosis” does not exclude the possibility ofobtaining treatment (e.g., simultaneous or not simultaneous) of bothdisorders.

Compositions Comprising Boysenberry and the Associated Bioactivity ofthese Compositions

The inventors have found that consumption of a Boysenberry compositionreduces allergen-induced lung remodelling in a chronic model of asthma.For these experiments, the effect of Boysenberry consumption was testedon lung fibrosis, lung macrophage phenotype, and MMP-9 expression in achronic model of allergic airway inflammation. The results demonstratedthat oral Boysenberry treatment supports the development of lungmacrophages that express a mixed antifibrotic, AAM (alternativelyactivated macrophages) phenotype with the capacity to amelioratefibrosis and promote balanced lung repair (74; incorporated herein byreference in its entirety). Further to this, the inventors have foundthat combined administration of Boysenberry and apple compositions, andcombined administration of Boysenberry, apple and blackcurrantcompositions, can be used to reduce numbers of immune cells associatedwith airways inflammation. This can include one or more of eosinophils,neutrophils, monocytes, and antigen presenting cells. Surprisingly, andadvantageously, the combined Boysenberry and apple compositions areeffective at low dosages.

Boysenberries are known to be high in Vitamin C and fibre and containhigh levels of anthocyanins (120-160 mg/100 g) that give Boysenberriestheir deep, dark colour. The ORAC (oxygen radical absorption capacity,i.e., antioxidant level) for Boysenberries is 42 μmoles/TE/g almostdouble that of blueberries, a well known antioxidant food. Boysenberriescontain notable amounts of ellagic acid, a phenolic compound. Theellagic acid level in Boysenberries is 5.98 mg/g of dry weight.Boysenberries also have a high ratio of free ellagic acid to totalellagitannins. The inventors have tested concentrates to confirmbiological activity.

As described in Example 2, these solutions included: Boysenberry 10(Boysenberry juice solution), 6.7%, Boysenberry 1 (Boysenberry juicesolution), 0.67%, apple 10 (apple juice solution), 18.7%, apple 1 (applejuice solution), Boysenberry and apple 10 (BerriQi™ Boysenberry withapple; combined juice solution), 6.7%/18.7%, Boysenberry and apple 1(BerriQi™ Boysenberry with apple; combined juice solution), 0.67%/1.87%.Additional oral compositions are described in Examples 3-5, includingBerriQi™ Boysenberry with apple composition at 100%, 50% or 25%concentration, and BerriQi™ Boysenberry with apple and blackcurrantcomposition at 100% concentration. The 100% BerriQi™ Boysenberry withapple composition includes Boysenberry juice concentrate at 27% andapple juice concentrate at 73%. The 100% BerriQi™ Boysenberry with appleand blackcurrant composition includes Boysenberry juice concentrate at13.5%, apple juice concentrate at 7%, and blackcurrant juice concentrateat 13.5%. Further details are provided in the Examples section, below.

From the inventors' results it is evident that Boysenberries and applesand Boysenberry, apple and blackcurrant may be used in compositions fortreating or preventing inflammation of the respiratory tract, treatingor preventing asthma, treating or preventing chronic obstructivepulmonary disease, treating or preventing allergic airways inflammation,treating or preventing fibrosis of the respiratory tract, or treating orpreventing airway remodelling. In particular, as to airway remodelling,the compositions of the invention may be used to treat or prevent one ormore of: the thickening of the walls of the alveoli, increasing ofcollagen fibres in the airway, spreading of collagen fibres into theairway tissues, and collapsing or closing of the airspace(s). Inaddition, the compositions of the invention may be used to reduce tissueinflammation and collagen deposition that leads to airway remodelling.

In addition, from the results shown herein, it will be understood thatBoysenberry and apple compositions and Boysenberry, apple andblackcurrant compositions may be used to restore, improve, or maintainthe health of the respiratory system, for example, in one or moreactivities of: decreasing collagen deposition, abrogating aberrantcollagen deposition, decreasing cellular infiltration into the airways,decreasing airway damage due to cellular infiltration, reducing cells inthe lung fluid, e.g., inflammatory cells, reducing mucus production,reducing mucus-positive cells, decreasing hydroxyproline levels,increasing matrix metallopeptidase expression levels, e.g., proteinlevels, increasing MMP-9 expression levels, e.g., protein levels,increasing TGFβ expression levels, e.g., protein levels, decreasing theratio level of TIMP-1/MMP-9, e.g., protein ratio levels, decreasing theactivation or number of inflammatory cells, increasing the number oractivity of alternatively activated macrophages, increasing the numberor activity of arginase+macrophages, increasing the number or activityof CD68+/CD206+/arginase+macrophages, or decreasing iNOS expressionlevels, e.g., protein levels. Further uses for these compositions aredescribed in detail herein.

Methods of Producing Compositions Comprising Boysenberry and Apple andCompositions Comprising Boysenberry, Apple and Blackcurrant

The present invention relates generally to a composition prepared fromBoysenberry and apple, as well as a composition prepared fromBoysenberry, apple and blackcurrant. While specific combinations ofBoysenberry and apple, and Boysenberry, apple and blackcurrant, aredescribed herein, it is understood that the composition of the inventioncan include any combination of Boysenberry, apple, and blackcurranttherein. In one particular aspect, the composition is prepared fromRubus ursinus var loganobaccus cv Boysenberry. In other aspects, one ormore genetic derivatives from this Boysenberry plant may be used. Forexample, it may be desirable to use F1 or F2 progeny from a geneticcross that includes the parent stock of the Boysenberry plant.Alternatively, any sports or other cultivars obtained from the parentmay be used. It may be desirable to source the Boysenberries from NewZealand, in particular, or alternatively, from Chile.

The composition is preferably prepared as a Boysenberry concentrate, forexample, a Boysenberry puree, a Boysenberry pomace, Boysenberry paste,Boysenberry powder, Boysenberry juice concentrate. The Boysenberry andapple composition may comprise an apple concentrate, for example, anapple puree, an apple pomace, an apple paste, an apple powder, or applejuice concentrate. If an apple juice concentrate is used, this may haveoriginated from cloudy apple juice or clear apple juice. TheBoysenberry, apple and blackcurrant composition may comprise ablackcurrant concentrate, for example, a blackcurrant puree, ablackcurrant pomace, a blackcurrant paste, a blackcurrant powder, ablackcurrant juice concentrate. It will be understood that Boysenberry,apple, or blackcurrant pomaces, in particular, may be used, whichinclude the solid remains of the fruit after pressing for juice. Thepomace may encompass one or more of the skins, pulp, seeds, and stems ofthe fruit. In addition, it is possible to include one or more of aBoysenberry juice, an apple juice, and a blackcurrant juice in thecompositions disclosed herein.

Accordingly, the composition may be prepared in liquid or powdered form,for example, a lyophilised powder, or in any other suitable dosage form.The composition may be formulated as a tonic, extract, elixir, linctus,concentrate, syrup, solution, suspension, emulsion, draught, puree,paste, or as drops. In other aspects, the composition may be formulatedas a gel or jelly, or a capsule, for example, with liquid or semi-liquidcontents. The composition may be provided in sachet form, for example, apowder sachet, or a gel or jelly sachet. Included also are formulationscomprising thin strips, or comprising solids in a capsule to mix withfood or drink. Other formulas are also possible, as described hereinbelow.

In certain aspects, it may be desirable to formulate the Boysenberry andapple composition (e.g., Boysenberry and apple juice concentrate orpuree) or the Boysenberry, apple and blackcurrant composition (e.g.,Boysenberry, apple and blackcurrant juice concentrate or puree) into apowder. As specific exemplifications, apple powder may comprise applepomace powder or apple pectin powder. Commercial Boysenberry, apple, andblackcurrant powders are known and available, as noted herein. Thepowder may be formulated as tablets (including rapid dissolve tablets)or capsules (including extended release capsules). The tablets may bescored tablets, chewable tablets, effervescent tablets, orallydisintegrating tablets, or tablets for forming a suspension. Thecapsules may be gel capsules, for example, and may include powderedcontents. This includes gel capsules made by single piece gelencapsulation and two piece gel encapsulation. Non-gelatine capsules arealso included, as well as caplets. The powder may be provided in freeflowing form or as a solid cake. The composition may be provided as apowder for forming a suspension, powder for forming a solution, bulkoral granules, or bulk oral powder.

The compositions of the invention may be prepared from Boysenberry,apple, or blackcurrant juice concentrate or puree obtained from one ormore commercial sources. For example, commercial sources of New ZealandBoysenberry products include Boysenberries New Zealand Ltd, Nelson, andTasman Bay Berries, Nelson. Commercially available products includeindividually quick frozen berries, Boysenberry puree, block frozenberries, Boysenberry juice concentrate, and Boysenberry powder.Commercial sources of New Zealand apple and blackcurrant productsinclude those from EnzaFoods New Zealand Ltd, Hastings, New Zealand, aswell as Juice Products New Zealand, Timaru, New Zealand, NZ BlackcurrantCo-operative Ltd, Nelson, New Zealand, Fruit Solutions, FSL Foods,Nelson, New Zealand, and Reso, Auckland, New Zealand, and encompasscloudy apple juice concentrate, clear apple juice concentrate, applepomace, apple puree, blackcurrant juice concentrate, and blackcurrantpuree. In addition, apple and blackcurrant powders may be obtainedcommercially from TreeTop®, FutureCeuticals, Nutradry, Sujon, Viberi™,Zeaberry™, Waitaki Biosciences, amongst other suppliers. Specificsuppliers of apple and blackcurrant juice concentrate include InfruitLtd, Auckland, New Zealand, and RD2 International Ltd, Auckland, NewZealand, while manufacturers include Profruit (2006) Ltd, Hastings,4175, New Zealand, as noted herein.

The pH of the juice concentrate or puree, for example, the combinedBoysenberry and apple juice concentrate, and combined Boysenberry, appleand blackcurrant juice concentrate, may range from 3.2 to 3.8; or 3.0 to4.0; or 3.1 to 3.9; or may be about 3.1, about 3.2, about 3.3, about3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about4.0. For the apple juice concentrate that is used to make the combinedconcentrate, the pH may range from 2.8 to 4.4; 2.9 to 4.3; 3.0 to 4.2;or 3.1 to 4.0; or may be about 2.8, about 2.9, about 3.0, about 3.1,about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4,or about 4.5. For the blackcurrant juice concentrate that is used tomake the combined concentrate, the pH may range from 1.0 to 5.0; 2.0 to4.0; 1.5 to 3.5; 2.1 to 3.4; or 2.3 to 3.3; or may be about 1.8, about1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5,about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8,about 3.9, or about 4.0.

For the Boysenberry juice concentrate, the acidity (% w/w citric acidanhydrous) may be about 1 to about 20, about 1.5 to about 15, about 2 toabout 12, about 5 to about 10, about 6 to about 9, about 10, about 9,about 8.5, about 8.3, about 8.2, about 8.17, about 8.1, about 8, about7, about 6, or about 5. For the apple juice concentrate, the acidity (%w/w malic) may be about 0.5 to about 4.5, about 0.8 to about 4.2, about1.0 to about 4.0, about 1.2 to about 3.5, or about 0.5, about 0.7, about0.9, about 1, about 1.2, about 1.5, about 1.7, about 1.9, about 2, about2.2, about 2.5, about 2.7, about 2.9, about 3, about 3.2, about 3.5,about 3.7, about 3.9, about 4, about 4.2, or about 4.5. For theblackcurrant juice concentrate, the acidity (citric acid g/100 g) may beabout 5 to about 20, about 8 to about 18, about 7 to about 17, or about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, or about 25.

In some circumstances, it may be desirable to adjust the pH of the pureeor that of the final composition to approximate physiological levels. Inparticular, it may be useful to obtain a pH range from 6.0 to 8.0; or6.5 to 7.5; or 6.8 to 7.2; or a pH of about 6.5, about 6.7, about 6.8,about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, orabout 7.5.

In certain aspects, the compositions of the invention may be prepared by“soft pulping” technology referred to in New Zealand Patent No. 235972(incorporated by reference herein), which can be adapted to produce a“soft” Boysenberry, apple, or blackcurrant puree. It may be useful toprepare the puree to have seeds removed. It may also be useful toprepare the puree with a sieve size of about 1 mm or less.

A juice concentrate may be prepared as a natural sugar solution that isextracted or pressed and filtered from the skin and pulp, and mayinclude the seeds. The solution may be depectinized, filtered, andevaporated under vacuum to a specified Brix level. For example, thejuice concentrate may be folded about two to about seven times theoriginal Brix value. In particular, the concentrate may be folded abouttwo times, about three times, about four times, about five times, aboutsix times, or about seven times the original Brix value.

In certain aspects, the Boysenberry juice concentrate may bemanufactured from sound, ripe graded boysenberries (e.g., Rubus ursinusvar loganobaccus cv Boysenberry). In particular aspects, the Boysenberryjuice concentrate may have a final sugar level ranging from 55° to 75°Brix; or 59° to 69° Brix; or 61° to 66° Brix; or about 60°, about 61°,about 62°, about 63°, about 64°, about 65°, about 65.4°, about 65.5°,about 65.6°, about 66°, about 67°, about 68°, about 69°, about 70°, orabout 71° Brix. In various aspects, the combined Boysenberry and applejuice concentrate, and the combined Boysenberry, apple and blackcurrantjuice concentrate may have the Brix values as noted directly above.Similarly, the apple juice concentrate and blackcurrant juiceconcentrate that is used to make the combined concentrate may have theBrix values as noted above, when uncorrected for acidity.

The juice concentrate may be produced by milling, mashing and pressinginto single strength juice which is centrifuged, pasteurized,depectinised, filtered and then concentrated by evaporation with aromareturned in the standardisation process. The standardised concentratemay then be packed through the hygienic filler head into the requiredpack style without further heat treatment. The concentrate can bechecked for compliance with the definition of a pure fruit juice, forexample, as defined by the FSANZ—Food Standards Australia New Zealand.

It is expected that the Boysenberry juice concentrate will be rich incolouration. For example, the Boysenberry juice concentrate may have acolour ratio (absorbance 520 nm/absorbance 430 nm) of about 1.5 to about3.0, about 1.8 to about 2.8, about 1.9 to about 2.2, or about 1.9, about2, about 2.01, about 2.05, about 2.1, or about 2.2. In addition, thejuice concentrate may have a colour intensity (utilising Chroma meter)of about 15 to about 30, about 20 to about 28, about 21 to about 25,about 22 to about 24, or about 22, about 23, about 23.2, about 23.5,about 23.7, about 24, or about 25. The juice concentrate is alsoexpected to be relatively clear in appearance, for example, with claritylevels of about 0.01 to about 0.1, about 0.02 to about 0.08, about 0.03to about 0.06, about 0.04 to about 0.05, or about 0.03, about 0.04,about 0.045, about 0.047, about 0.048, about 0.05, or about 0.06.

The blackcurrant concentrate that is used to make the combinedconcentrate will also have deep colouration. For example, theblackcurrant concentrate may have a colour ratio (absorbance 520nm/absorbance 430 nm at pH 3) of about 1.5 to about 4.0, about 1.8 toabout 3.8, or about 2.0 to about 3.0; or a colour ratio of about 1.8, orabout 1.9, or about 2.0, or about 2.1, or about 2.2, or about 2.3, orabout 2.4, or about 2.5, or about 2.6, or about 2.7, or about 2.9, orabout 3.0, or about 3.2, or about 3.3, or about 3.4, or about 3.5.

In contrast, the apple juice concentrate that is used to make thecombined concentrate may be relatively colourless, for example, lessthan 0.35 abs at 420 nm 12 Bx, or at least less than 0.45 abs at 420 nm12 Bx. For apple juice concentrate, this can also be express as ageneral range of about 0.15 to about 0.45 abs, about 0.10 to about 50abs, or about 0.19 to about 45 at 440 nm and 11.5° Brix. The apple juiceconcentrate may be used as a clear concentrate, e.g., free from haze.The specific gravity of the various juice concentrates may be about 1.2to about 1.4, about 1.29 to about 1.39, or about 1.32 to about 1.36, orabout 1.2, about 1.3, about 1.31, about 1.32, about 1.33, about 1.35,about 1.36, about 1.37, about 1.38, about 1.39, or about 1.4 at 20° C.The various measurement methodologies, e.g., colour ratios, clarity,etc, are known in the art, and may be found, for example, in the AIJNcode of practice in the International Fruit Juice Federation Handbook ofAnalysis, 1996, International Fruchtsaft-Union, Zug, Switzerland.

In initial preparatory stages, the Boysenberry, apple, or blackcurrantmay undergo a pre-treatment process which may include the well knownsteps of ripening, inspecting, grading, and/or sorting of theberries/fruit. With regard to ripening, it is preferable to use ripe ormature Boysenberry, apple, or blackcurrant when producing thecompositions of the invention; however, rotted or decaying material ispreferably avoided. Ripeness can be assessed using widely known and usedmethods in the art. Ripeness can be measured prior to picking orprocessing the Boysenberry, apple, or blackcurrant. In particular,ripeness may be measured using the Brix system, as noted herein.Boysenberry, apple, or blackcurrant that is overly mature or fermentingmay not produce an ideal composition. Boysenberry, apple, orblackcurrant with a Brix level below the ideal may be artificiallyripened before use.

As part of the processing, the Boysenberry, apple, or blackcurrant maybe sterilised. The fruit may be passed through an assembly having one ormore roller brushes for removing any adhering foreign matter.Conventional washing techniques may then be employed. For example, it ispossible to use a series of spray nozzles to wash the Boysenberry,apple, or blackcurrant. Wash additives aiding cleansing or reducing thebacteria count on the Boysenberry, apple, or blackcurrant may beemployed according to local regulations and requirements. For example,the Boysenberry, apple, or blackcurrant may be washed by a chlorine washand/or an ozone impregnated water wash followed by a fresh water rinse.

The sterilized Boysenberry, apple, or blackcurrant may then be conveyedinto a hopper. This can be tapered to form a funnel to direct theberries or fruit to a pressing assembly. The pressing assembly may beadapted to perform a pulping or comminution process. Such process can berelatively mild and gentle (“soft pulping”) compared to conventionalfruit pulping techniques. With soft pulping, no significantdisintegration or lysis of fruit cells or components. Preferably, only aminor proportion (generally less than 5-10%) of seeds is fragmented bythis process.

In one embodiment, the pressing assembly performs the soft pulping ofthe Boysenberry, apple, or blackcurrant by pressing between a twinconverging belt press. The press belts may be multiple loops rotatedabout a series of pulleys. The distance separating the press belts maydecrease in the direction of travel of the Boysenberry, apple, orblackcurrant. In this way, increased force may be exerted upon theBoysenberry, apple, or blackcurrant as it travels along the length ofthe pressing assembly. This can produce pulping without significantdamage to the seeds. This in turn prevents seeds from contaminating thepulp.

The pulp generated from the pressing assembly may be directed to ascreening process, in order to separate the seeds from the pulp. Inparticular, the pulp may be separated from the seed using a softmechanical screening technique. For example, a pulp finisher may beused. This includes a rotating flexible impeller which is rotated withina cone shaped screen having apertures of a predetermined size. Inparticular aspects, the size of the apertures is selected to permit thepulp and juice to pass through the screen while retaining a substantialportion, if not all, of the seeds within the interior cavity defined bythe screen.

In certain aspects, it may be preferable to use a paste rather than apuree from the Boysenberry, apple, or blackcurrant. A paste may be madeas a concentrate. For example, the fruit may be heated for severalhours, strained, and reduced to a thick, concentrated form. The fruitmay be heated after removing the skins, or after the pulping or pureeingprocess. The fruit can be heated gradually, and then kept heated at amoderate temperature, with mixing. Upon thickening, the paste can bespread on a flat sheet, or transferred to a packaging, for example, abag, tube, jar, bottle, or other container. The paste may be transferredaseptically, such that it is suitable for human consumption. It may bedesired to prepare the paste from mature berries/fruits. The paste maybe prepared from pulped fruit. The paste may be a smooth preparation.

The pulp (e.g., in paste or puree form) or juice concentrate may beprocessed by a freezing step. This may be followed by or used inconjunction with a drying step. In an alternative embodiment, the pulpis dried and processed to a powder without an intervening freezing step.For example, methods involving drum drying may be used. In thedrum-drying process, a puree or paste may be dried at relatively lowtemperatures over rotating, high-capacity drums that produce sheets ofdrum-dried product. In certain aspects, an additive may be used toaccelerate or otherwise assist the drying process. For example, peastarch or other drying aids may be utilised. The dried product may thenbe milled to a finished flake or powder form. Advantageously, drumdrying techniques may be used to produce a dried composition thatretains its key components, e.g., phenolic compounds, and can be easilyreconstituted using liquid. For example, drum dried products may be madeto be cold water soluble. As further alternatives, belt drying orconvection drying may be used. Such drying methods are widely known andused in the field.

If freezing is used, it is preferable to freeze the pulp or juiceconcentrate as soon as possible after it is produced to maintainfreshness. However, freezing may be carried out within 24 or 48 hours,as needed. Freezing methodologies are well known and need not bedescribed in significant detail herein. Blast freezing is particularlypreferred for use with the invention. The pulp or juice concentrate maybe frozen in standard sized pales, which are used to collect the freshpulp/concentrate after processing. The pulp or juice concentrate can bestored frozen (e.g., at −18° C.) until it is required.

The frozen pulp or juice concentrate may be freeze dried, i.e.,lyophilised. Freeze drying techniques are widely known and commonlyused. The freeze drying cycle may be about 48 hours; or ranging from 40to 56 hours; or 12 to 36 hours; or 36 to 60 hours; or about 40 hours,about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50hours, about 52 hours, or about 54 hours. A longer freeze drying cycle,e.g., at least 48 hours (“gentle freeze drying”), may be used to retainmaximal activity. In particular aspects, the process may be carried outto such that water formation is avoided, and the moisture content isminimised during processing.

It may be desirable to use a particular lyophilisation process forobtaining the dried product. For example, a lyophilisation dryingprogram may be used as part of an automated drying system. Thelyophilisation process may include multiple drying steps, e.g., withstep-wise increases and reductions in temperature. Preferably, a primarydrying setting is used for sublimation, followed by one or moresecondary drying settings that are used to remove residual moisture. Inparticular aspects, the top temperature of the lyophilisation processdoes not exceed 70° C. In other aspects, the temperature of thelyophilisation process ranges between −10° C. to 70° C. In one otheraspect, up to 48 hours of lyophilisation is utilised.

The resulting dried product may then be milled into a powder which canthen be utilised as appropriate. Milling methods are well known andwidely used in the art. Standard mesh sizes may be used to produce thepowder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50mesh sizes may be used. The sieve size for the powder may range from 1.0to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or may be about 1.0 mm,about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, orabout 0.3 mm.

To ensure minimal degradation of ingredients, the preparation processmay be performed at a temperature of less than 40° C. In variousembodiments, the process is performed at a temperature ranging from −4°C. to 40° C.; or from −1° C. to 10° C.; or from 1° C. to 6° C.; or atabout 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about5° C., or about 6° C. These temperatures may be kept during the entirepreparation process, including the storage of the whole fruit, prior toit being broken open, and during the pulping/pureeing process. Foroptimal results, these temperatures are kept at least from the pointthat the fruit has been broken open. Use of such temperatures avoidsoxidation of the fruit and the use of reducing agents. In certaincircumstances, it may be possible to obtain organic certification.

The processing method is preferably performed so as to prevent or atleast minimise any damage or effects on the active material in thefruit. To ensure optimal production methods, the resulting compositionscan be monitored for activity, for example, for anthocyanin levels,polyphenol levels, and/or antioxidant activity.

Assays for polyphenols are well known in the art and are also describedbelow. In particular, it is possible to measure gallic acid equivalents(GAE) to determine total polyphenol content. For example, theFolin-Ciocalteu method (employing the Folin-Ciocalteu reagent, alsocalled Folin's phenol reagent or Folin-Denis reagent) may be used forcolorimetric in vitro assays of phenolic compounds (75). It is expectedthat the total polyphenol content of a Boysenberry juice concentratewill be relatively high, for example, about 500 to about 5000 mg GAE/100g FW, about 1000 to about 3000 mg GAE/100 g FW, about 1500 to about 2500mg GAE/100 g FW, about 3000, about 2500, about 2000, about 1500, orabout 1000 mg GAE/100 g FW. It is noted that FW indicates the freshweight of the juice concentrate.

Anthocyanins may be quantified by HPLC. This can be used give breakdownof individual compounds and expressed as cyanidin 3-glucosideequivalents (76). For example, HPLC eluted components may be monitoredat 530 nm for anthocyanins. A standard curve may be prepared using acyanidin-3-glucoside standard (for example, from Extrasynthese) andtotal anthocyanins may be calculated on this basis. Other phenolics mayalso be analysed by HPLC, for example at 250-700 nm. A range ofstandards may be run, including gallic acid, ellagic acid, quercetin,rutin and catchin. Absorbance spectra and retention time of thestandards may be compared with unknowns in the HPLC traces. Thisanalysis can include measurements for ellagic acid. As non-limitingexamples, the total anthocyanin content of a Boysenberry juiceconcentrate (expressed as cyanidin 3-glucoside equivalents) may be about1000 to about 10,000 mg/100 g FW, about 2000 to about 8000 mg/100 g FW,about 4000 to about 7000 mg/100 g FW, about 5500 to about 6500 mg/100 gFW, or about 8000, about 7000, about 6500, about 6800, about 6000, about5000, about 4000, or about 3000 mg/100 g FW.

For the combined Boysenberry and apple compositions, it is expected thatthe total anthocyanins may account for about 40-50% of the totalpolyphenol content that is present in these compositions, or at least40%, at least 41%, at least 42%, at least 43%, at least 44%, at least45%, at least 46%, at least 47%, at least 48%, at least 49%, at least50%, at least 51%, at least 52%, at least 53%, at least 54%, or at least55% of the total polyphenol content that is present in thesecompositions. For the combined and Boysenberry, apple and blackcurrantcompositions, it is expected that the total anthocyanins may account forabout 70-80% of the total polyphenol content that is present in thesecompositions, or at least 65%, at least 66%, at least 67%, at least 68%,at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, or at least 80%, of the total polyphenol content that ispresent in these compositions.

In addition, for the combined Boysenberry and apple compositions and theBoysenberry, apple and blackcurrant compositions, it is expected thatthe total polyphenols (including anthocyanins) may account for about80-90% of the total polyphenol content that is present in thesecompositions, or at least 70%, at least 71%, at least 72%, at least 73%,at least at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, or at least 95% of the total polyphenol contentthat is present in these compositions.

Further to this, for the combined Boysenberry and apple compositions andthe Boysenberry, apple and blackcurrant compositions, it is expectedthat the hydrolysable tannins will account for about 25% to about 35% ofthe total polyphenols in the composition. For the Boysenberry, apple andblackcurrant compositions, it is expected that the hydrolysable tanninswill account for about 8% to about 12% of the total polyphenols in thecomposition. An exemplary method for measuring hydrolysable tannins isLC-MS (liquid chromatography-mass spectrometry) analysis, as describedin detail herein.

Antioxidant capacity may be measured by ORAC and/or DPPH assays. Theoxygen radical absorbance capacity assay is one of the most widelyutilised assays to test the antioxidant potential of foods. The ORACassay measures antioxidant inhibition of peroxyl radical-inducedoxidation (77, 78, 84). Trolox, a water-soluble analogue of vitamin E,may be used as a control standard. In an additional assay, DPPH(2,2-diphenyl-1-picrylhydrazyl) may be used to show the kineticbehaviour of polyphenols as free radical scavengers. The higher theantioxidant activity, the larger the decrease of DPPH⋅ concentration. Amethanolic solution of the DPPH radical changes from purple tocolourless when quenched by antioxidants. The decrease in DPPH⋅ ismeasured at 515 nm against standard curves, e.g., Trolox and DPPH⋅ (79,80).

As particular exemplifications, the antioxidant capacity for theBoysenberry juice concentrate may be about 10,000 to about 100,000 ORACvalue (μmol Trolox/100 g FW), about 20,000 to about 80,000 ORAC value,about 30,000 to about 70,000 ORAC value, about 40,000 to about 50,000ORAC value, or about 80,000, about 70,000, about 60,000, about 50,000,about 40,000, about 30,000, or about 20,000 ORAC value. As furtherexemplifications, the antioxidant capacity for the Boysenberry juiceconcentrate may be measured with the DPPH assay (at 100% MeOH) as about1000 to about 6000 μmol TEAC/100 g FW, about 2000 to about 5000 μmolTEAC/100 g FW, about 2500 to about 2900 μmol TEAC/100 g FW, or about5000, about 4000, about 3000, about 2800, about 2500, about 2000, orabout 1000 μmol TEAC/100 g FW.

Alternatively or additionally, the compositions can be tested for othercomponents, e.g., sugars, folate, and Vitamin C. The correspondingassays are widely known. For example, folate levels of the Boysenberryjuice concentrate may be measured using standard methodologies (see,e.g., 83), and may be about 20 μg/100 g FW, about 30 μg/100 g FW, about40 μg/100 g FW, or about 50 μg/100 g FW, about 60 μg/100 g FW, about 70μg/100 g FW, or about 80 μg/100 g FW, or about 10 to about 100 μg/100 gFW, about 20 to about 80 μg/100 g FW, about 30 to about 70 μg/100 g FW,about 20 to about 50 μg/100 g FW, or about 50 to about 70 μg/100 g FW.

It will be understood that other known assays may also be used toanalyse the disclosed compositions (see, e.g., 85), and the invention isnot limited to one particular assay for bioactive compounds, includingphenolics, anthocyanins, antioxidants, vitamins, carbohydrates, etc. Itwill be understood also that the levels identified herein for juiceconcentrates can be readily extrapolated to powdered forms, as well aspuree and paste forms.

In some circumstances, it may be possible to use genetic derivative ofthe plant to obtain the compositions of the invention. It is expectedthat a composition obtained from such derivative would share one or moreof the characteristics of the compositions obtained from the originalstock. Exemplary features include: polyphenol levels and polyphenolprofiles, including anthocyanidin levels and profiles, vitamin levels,and reduction of OVA-induced inflammation, as noted above and disclosedin detail herein. Regarding the fruit itself, it is expected that thefruit obtained from a genetic derivative would share a similarcompositional makeup as the parent fruit.

Compositions Comprising Boysenberry and Apple and CompositionsComprising Boysenberry, Apple and Blackcurrant

The inventors have found that Boysenberry compositions includebeneficial ingredients that are useful for maintaining the health of therespiratory system, as well as treating and preventing respiratoryproblems. The inventors have shown that a Boysenberry concentrate isparticularly efficacious for reducing airway inflammation and fibrosis.Also efficacious are a combined Boysenberry and apple concentrate, and acombined Boysenberry, apple and blackcurrant concentrate, as describedherein. As such, the Boysenberry compositions, including combinedBoysenberry and apple compositions, and combined Boysenberry, apple andblackcurrant compositions, disclosed herein can be used to support orimprove overall respiratory health and/or to treat or prevent variousdisorders or other conditions of the respiratory tract, includinginflammation, asthma, chronic obstructive pulmonary disease, airwayfibrosis, and airway remodelling. In this way, the disclosedcompositions are understood to be anti-inflammatory compositions, andalso anti-asthmatic compositions, as well as being compositions that areactive against chronic obstructive pulmonary disease, reactive airwaydisease, airway fibrosis, and airway remodelling.

As described herein, a Boysenberry composition may comprise a juiceconcentrate or a powder concentrate prepared from Boysenberries. Thecomposition may further comprise a juice concentrate or a powderconcentrate prepared from apples, or may further comprise a juiceconcentrate or a powder concentrate prepared from blackcurrants. Asvarious alternatives, the composition may consist of, or may consistessentially of: a juice concentrate or a powder concentrate preparedfrom Boysenberries and a juice concentrate or a powder concentrateprepared from apples, or a juice concentrate or a powder concentrateprepared from Boysenberries and a juice concentrate or a powderconcentrate prepared from blackcurrants.

Generally speaking, the Boysenberry and apple concentrate may includevarious ratios of Boysenberry concentrate to apple concentrate. Asexemplifications, the percentages of Boysenberry concentrate to appleconcentrate (having a combined percentage of 100% v/v) may include about17% Boysenberry concentrate to about 83% apple concentrate, about 18%Boysenberry concentrate to about 82% apple concentrate, about 19%Boysenberry concentrate to about 81% apple concentrate, about 20%Boysenberry concentrate to about 80% apple concentrate, about 21%Boysenberry concentrate to about 79% apple concentrate, about 22%Boysenberry concentrate to about 78% apple concentrate, about 23%Boysenberry concentrate to about 77% apple concentrate, about 24%Boysenberry concentrate to about 76% apple concentrate, about 25%Boysenberry concentrate to about 75% apple concentrate, about 26%Boysenberry concentrate to about 74% apple concentrate, about 27%Boysenberry concentrate to about 73% apple concentrate, about 28%Boysenberry concentrate to about 72% apple concentrate, about 29%Boysenberry concentrate to about 71% apple concentrate, about 30%Boysenberry concentrate to about 70% apple concentrate, about 31%Boysenberry concentrate to about 69% apple concentrate, about 32%Boysenberry concentrate to about 68% apple concentrate, about 33%Boysenberry concentrate to about 67% apple concentrate, or about 34%Boysenberry concentrate to about 66% apple concentrate, thesepercentages being representative of v/v values.

In the same way, the percentages of Boysenberry and blackcurrantconcentrate to apple concentrate (having a combined percentage of 100%v/v) may include about 17% Boysenberry and blackcurrant concentrate toabout 83% apple concentrate, about 18% Boysenberry and blackcurrantconcentrate to about 82% apple concentrate, about 19% Boysenberry andblackcurrant concentrate to about 81% apple concentrate, about 20%Boysenberry and blackcurrant concentrate to about 80% apple concentrate,about 21% Boysenberry and blackcurrant concentrate to about 79% appleconcentrate, about 22% Boysenberry and blackcurrant concentrate to about78% apple concentrate, about 23% Boysenberry and blackcurrantconcentrate to about 77% apple concentrate, about 24% Boysenberry andblackcurrant concentrate to about 76% apple concentrate, about 25%Boysenberry and blackcurrant concentrate to about 75% apple concentrate,about 26% Boysenberry and blackcurrant concentrate to about 74% appleconcentrate, about 27% Boysenberry and blackcurrant concentrate to about73% apple concentrate, about 28% Boysenberry and blackcurrantconcentrate to about 72% apple concentrate, about 29% Boysenberry andblackcurrant concentrate to about 71% apple concentrate, about 30%Boysenberry and blackcurrant concentrate to about 70% apple concentrate,about 31% Boysenberry and blackcurrant concentrate to about 69% appleconcentrate, about 32% Boysenberry and blackcurrant concentrate to about68% apple concentrate, about 33% Boysenberry and blackcurrantconcentrate to about 67% apple concentrate, or about 34% Boysenberry andblackcurrant concentrate to about 66% apple concentrate, thesepercentages being representative of v/v values.

As non-limiting examples, the percentage of blackcurrant concentrate inthe combined Boysenberry, apple and blackcurrant concentrate may beabout 5% to about 20%, or about 8% to about 18%, or about 10% to about15%, or a percentage of about 5%, about 8%, about 10%, about 13.5%,about 15%, about 18%, or about 20%, these percentages beingrepresentative of v/v values. In particular aspects, the percentage ofthe blackcurrant concentrate is the same or substantially the same asthe percentage of Boysenberry concentrate in the combined Boysenberry,apple and blackcurrant concentrate.

The Boysenberry and apple concentrate and the Boysenberry, apple andblackcurrant concentrate (having a combined percentage of 100% w/v) mayinclude less than 1% of a preservative, for example, about 0.005% toabout 0.5%, or about 0.05% to about 0.15%, or may include about 0.04%,about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about0.16%, about 0.18%, or about 0.2% of a preservative, these percentagesbeing representative of w/v values. Useful preservatives include but arenot limited to sorbic acid, sodium sorbate, potassium sorbate, citricacid, ascorbic acid, malic acid, tartaric acid, propionic acid, andbenzoic acid, for example, in the form of its sodium salt, e.g., sodiumbenzoate.

The composition may be formulated as a liquid, for example, a juiceconcentrate, syrup, suspension, or tonic for oral administration, or asa solution for enteral administration. Alternatively, the compositionmay be formulated as a powder to be encapsulated, tableted, or added toor incorporated in other products. Particularly encompassed are delayedrelease formulas, extended release formulas, as well as formulas forrapid disintegration. Capsules, for example gel capsules, arespecifically encompassed, as well as sachets and chewable tablets.Additionally included are combination formulas, which include the powderof the invention mixed with other beneficial agents, e.g., one or morerespiratory aids. In various aspects, the composition may be prepared asa nutraceutical composition, a pharmaceutical composition, a functionalfood or beverage, a natural ingredient (e.g., a natural additive), or anatural supplement (e.g., a dietary supplement).

It is expected that the Boysenberry composition, including the combinedBoysenberry and apple composition and the combined Boysenberry, appleand blackcurrant composition, will be prepared to include high levels ofanthocyanins. For example, the composition may include about 2 to about50,000 mg/ml total anthocyanins or total Boysenberry and blackcurrantanthocyanins, or about 20 to about 40,000 mg/ml, about 25 to about35,000 mg/ml, about 30 to about 30,000 mg/ml, about 40 to about 25,000mg/ml, about 50 to about 20,000 mg/ml, about 60 to about 15,000 mg/ml,about 70 to about 10,000 mg/ml, about 80 to about 8000 mg/ml, about 90to about 6000 mg/ml, about 100 to about 5000 mg/ml, about 10 to about1000 mg/ml, about 20 to about 800 mg/ml, about 30 to about 600 mg/ml,about 50 to about 200 mg/ml, or about 50,000, about 40,000, about35,000, about 25,000, about 20,000, about 15,000, about 12,000, about10,000, about 8000, about 7500, about 5000, about 2500, about 2000,about 1000, about 1500, about 1200, about 1000, about 750, about 500,about 250 mg/ml, about 200 mg/ml, about 150 mg/ml, about 100 mg/ml,about 75 mg/ml, about 50 mg/ml, about 25 mg/ml, about 20 mg/ml, or about10 mg/ml total anthocyanins, or total Boysenberry and blackcurrantanthocyanins, or a dry weight equivalent thereof.

In specific aspects, the Boysenberry composition, including the combinedBoysenberry and apple composition and the combined Boysenberry, appleand blackcurrant composition, may be administered at a dosage unit ofabout 1 mg to about 20,000 mg total anthocyanins or total Boysenberryand blackcurrant anthocyanins, or about 1 mg to about 2000 mg totalanthocyanins or total Boysenberry and blackcurrant anthocyanins, orabout 5 mg to about 5000 mg, about 10 mg to about 3000 mg, about 10 toabout 1000, about 15 mg to about 1500 mg, about 20 mg to about 1000 mg,about 25 mg to about 850 mg, about 30 mg to about 600 mg, about 35 mg toabout 550 mg, about 50 to about 500 mg, about 5 to about 500, about 10mg to about 200 mg, about 1 to about 400 mg, about 1 to about 200 mg,about 40 mg to about 400 mg, about 40 to about 200 mg, about 20 mg toabout 80 mg, about 30 mg to about 60 mg, about 45 mg to about 55 mg, orabout 20,000 mg, about 15,000 mg, about 12,000 mg, about 10,000 mg,about 7500 mg, about 5000 mg, about 4000 mg, about 3000 mg, about 2000mg, about 1500 mg, about 1200 mg, about 1000 mg, about or about 500 mg,about 400 mg, about 300 mg, about 200 mg, about 100 mg, about 90 mg,about 95 mg, about 80 mg, about 75 mg, about 70 mg, about 65 mg, about60 mg, about 55 mg, about 50 mg, about 45 mg, about 40 mg, about 35 mg,about 30 mg, about 25 mg, about 20 mg, or about 10 mg total anthocyaninsor total Boysenberry and blackcurrant anthocyanins. In particularaspects, the dosage unit may be about 50 mg to about 500 mg totalanthocyanins or total Boysenberry and blackcurrant anthocyanins.

The dosage units as noted above may be administered once per day, twiceper day, or three times per day, or more as needed. An exemplary, andnon-limiting, daily dosage may be about 10 mg to about 1000 mg totalanthocyanins or total Boysenberry and blackcurrant anthocyanins. Thedosage may be adjusted for pediatric, geriatric, overweight,underweight, or other patients, where required.

If a Boysenberry juice, apple juice, or blackcurrant juice concentrateis made by standard commercial production methods (large or smallscale), or obtained from commercial sources, the juice concentrate,including the combined Boysenberry and apple juice concentrates and thecombined Boysenberry, apple and blackcurrant juice concentrates, may beadministered at a dosage unit of about 0.5 to about 50 ml, about 0.5 toabout 20 ml, about 0.5 to about 10 ml, about 1 to about 9 ml, about 2 toabout 8 ml, about 3 to about 7 ml, about 4 to about 6 ml, or about 50,about 40, about 30, about 20, about 15, about 12.5, about 10, about 9,about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1,or about 0.5 ml of juice concentrate. In particular aspects, the dosageunit may be about 5 ml of juice concentrate. The various dosage unitsmay be administered once per day, twice per day, or three times per day,or more as needed. Dosage modification can be made for patient size andage in accordance with known methods.

Each of the Boysenberry, apple, and blackcurrant concentrates will berich sources of phenolics, anthocyanins, and other beneficialcomponents. For example, a blackcurrant juice concentrate will beexpected to include a total anthocyanin content (g/100 g) of at least0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least1.3, at least 1.4, or at least 1.5. For exemplary anthocyaninquantitation methods, see (98) and (99). The blackcurrant juiceconcentrate will also be expected to include Vitamin C (ascorbic acid;mg/100 g) levels of at least 500, at least 550, at least 600, at least650, at least 700, at least 750, at least 780, at least 800, at least850, or at least 900. The blackcurrant juice concentrate will further beexpected to include a total phenolics content (g/100 g) of at least 3.0,at least 3.2, at least 3.4, at least 3.6, at least 3.8, at least 4.0, atleast 4.2, at least 4.4, at least 4.6, at least 4.8, or at least 5.0.For exemplary phenolics quantitation methods, see (100).

In certain circumstances, it may be desirable to isolate or enrich thepolyphenols from the fruits being used. In particular, it may beadvantageous to use the Boysenberry, apple, or blackcurrant to obtainpolyphenol enriched compositions, phenolic concentrates, or compositionscomprising isolated phenolics, e.g., isolated anthocyanins. For example,the compositions of the invention may be enriched for polyphenols suchthat their concentration is increased relative to the other componentsof the fruit, e.g., sugars. In particular aspects, the compositions ofthe invention may include polyphenols that have been isolated away from(e.g., purified from) the other components of the fruit. The particularpolyphenols for isolation or enrichment are described in detail herein.

Methods of enriching and extracting polyphenols are widely known in theart (e.g., 81, 82). The resulting composition may include at least 2times, at least 3 times, at least 4 times, at least 5 times, or at least10 times the amount of polyphenols compared to the composition preparedwithout polyphenol enrichment or isolation steps. The polyphenolenriched compositions, phenolic concentrates, and compositionscomprising isolated phenolics may be dried as a powder, and used inaccordance with the present invention.

The dosage form may contain excipients, for example, one or moreanti-adherents, binders, coatings, disintegrants, flavours, colours,sweeteners, lubricants, glidants, flow agents, anti-caking agents,sorbents, or preservatives. Useful excipients include but are notlimited to: stearin, magnesium stearate, and stearic acid; saccharidesand their derivatives, e.g., disaccharides: sucrose, lactose;polysaccharides and their derivatives, e.g., starches, cellulose ormodified cellulose such as microcrystalline cellulose and celluloseethers such as hydroxypropyl cellulose; sugar alcohols such as isomalt,xylitol, sorbitol and maltitol; proteins such as gelatin; syntheticpolymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids,waxes, shellac, plastics, and plant fibres, e.g., corn protein zein;hydroxypropyl methylcellulose; crosslinked polymers, e.g., crosslinkedpolyvinylpyrrolidone (crospovidone), and crosslinked sodiumcarboxymethyl cellulose (croscarmellose sodium); sodium starchglycolate; silicon dioxide, fumed silica, talc, and magnesium carbonate.

It is expected that the Boysenberry compositions disclosed herein willinclude various components, for example, carbohydrates and polyphenols,and in particular, anthocyanidins. Anthocyanidins of interest includecyanidins and rutinosides, such as cyanidin-3-O-sophoroside,cyanidin-3-O-glucoside, epicatechin, cyanidin-3-O-glucosylrutinoside,cyanidin-3-O-rutinoside,cyanidin-3-(6′-p-coumaryl)glycoside-5-glycoside, cyanidin-3-O-glycoside,cyanidin-3,5-diglycoside, and cyanidin-3-O-2G-glucosylrutinoside. Alsoof interest are hydrolysable tannins such as ellagitannins and ellagicacid. The Boysenberry compositions of the invention may also includevarious carbohydrates, and in particular, various sugars, includingneutral sugars. As to neutral sugars, the Boysenberry compositions mayinclude one or more of fructose and glucose, as well as sucrose.

Similarly, the apple compositions as disclosed herein will encompassvarious components, including various carbohydrates and polyphenols. Ofparticular interest are phenolic compounds such as flavonoids andcinnamic and benzoic acid derivatives. Included are catechins,procyanidins, and hydroxycinnamates, and more particularly included areincluded are 3-hydroxy flavonoids such as anthocyanins, flavanols, andflavan-3-ols. Key individual compounds include epicatechin, catechin,procyanidin dimer B2, 5-caffeoylquinic acid, and quercetin glycosides.Dihydrochalcones such as phloridzin are particularly included asflavonoid precursors found in apples. Apple peels can be used to obtainhigh levels of polyphenols and flavonoids such as quercetin glycosidesand cyanidin. Apple flesh and cores can be used to obtain high levels ofchlorogenic acid. Particular cultivars can be used to maximisepolyphenol levels. In particular, cider apples can be used to maximiseprocyanidins that are responsible for their astringency and bitterness.The apple compositions of the invention may also include variouscarbohydrates, and in particular, various sugars, including neutralsugars. As to neutral sugars, the apple compositions may include one ormore of fructose, glucose, and sucrose.

As disclosed herein, the blackcurrant compositions of the invention willinclude various components, including sugars and polyphenols. Notedspecifically are hydroxycinnamates such as chlorogenic andp-coumaroylquinic acids, and also anthocyanins, and flavonol glycosides.Of particular interest are polyphenol compounds such as delphinidin3-O-glucoside; delphinidin 3-O-rutinoside;delphinidin-3-O-(6-p-coumaroyl)glucoside; cyanidin 3-O-glucoside;cyanidin 3-O-rutinoside; cyanidin 3-O-glucoside-6-p-cumaryl; peonidin3-O-rutinoside; malvidin 3-O-rutinoside; neochlorogenic acid; p-coumaricacid glucoside; myricetin derivatives; quercetin 3-O-rutinoside;quercetin 3-O-galactoside; and quercetin 3-O-glucoside. For example, theskin may be used to obtain high levels of anthocyanins. The blackcurrantcompositions of the invention may also include various carbohydrates,and in particular, various sugars, including neutral sugars. As toneutral sugars, the compositions in the invention may include one ormore of fructose, glucose, and sucrose.

Methods of Using Compositions Comprising Boysenberry and Apple andCompositions Comprising Boysenberry, Apple and Blackcurrant

As noted above, the Boysenberry compositions disclosed herein, includingthe compositions comprising Boysenberry and apple, and the compositionscomprising Boysenberry, apple and blackcurrant, can be used to supportor improve overall respiratory health and/or to treat or prevent variousconditions of the respiratory tract, including inflammation, andrespiratory disorders associated with inflammation, such as asthma,chronic obstructive pulmonary disease, reactive airway disease, airwayfibrosis, and airway remodelling. Other conditions associated withinflammation in the respiratory tract include: allergy or allergicdisorders, emphysema, bronchitis, respiratory bronchiolitis,interstitial lung disease, inflammatory airway disease, fibrosingalveolitis, intrinsic alveolitis, pulmonary eosinophilia, pulmonaryvasculitis, pneumonia, interstitial pneumonia, desquamative interstitialpneumonia, lymphoid interstitial pneumonia, nonspecific interstitialpneumonia, eosinophilic pneumonia, pneumonitis, pleurisy (pleuritus),pleural effusion, cystic fibrosis, primary ciliary dyskinesia, acuterespiratory distress syndrome (ARDS), sarcoidosis, dermatomyositis,toxocariasis, Wegener's granulomatosis, Langerhans cell histiocytosis,Sjogren's syndrome, Kartagener syndrome, vocal cord dysfunction,spasmodic croup, autoimmune disease such as lupus, reflexive vasomotordisease, and autonomic disorders. Additional factors associated withinflammation in the respiratory tract include smoking, air pollution,allergens, infection (e.g., viral or bacterial), certain medication(e.g., chemotherapeutic agents), radiation treatment, medical devices(e.g., ventilators), and surgery.

The compositions of the invention find use for treating or preventingrespiratory tract inflammation, asthma, chronic obstructive pulmonarydisease, airway fibrosis, airway remodelling, or other conditionsdescribed herein. As exemplary dosages, the compositions may beadministered at dosages to obtain about 0.1 to about 200 mg/kg, about0.2 to about 180 mg/kg, about 0.25 to about 150 mg/kg, about 0.5 toabout 125 mg/kg, about 0.6 to about 100 mg/kg, about 0.7 to about 90mg/kg, about 0.1 to about 50 mg/kg, about 0.1 to about 20 kg/mg, about0.1 to about 10 mg/kg, about 0.1 to about 5 mg/kg, about 0.1 to about 1mg/kg, about 1 to about 20 mg/kg, about 1 to about 10 mg/kg, 1 to about5 mg/kg, or about 200 mg/kg, about 100 mg/kg, about 90 mg/kg, about 80mg/kg, about 70 mg/kg, about 60 mg/kg, about 50 mg/kg, about 40 mg/kg,about 30 mg/kg, about 20 mg/kg, about 10 mg/kg, about 9 mg/kg, about 8mg/kg, about 7 mg/kg, about 6 mg/kg, about 5 mg/kg, about 4 mg/kg, about3 mg/kg, about 2 mg/kg, about 1 mg/kg, about 0.9 mg/kg, about 0.8 mg/kg,about 0.7 mg/kg, about 0.6 mg/kg, about 0.5 mg/kg, about 0.4 mg/kg,about 0.3 mg/kg, about 0.2 mg/kg, or about 0.1 mg/kg, of totalanthocyanins or total Boysenberry anthocyanins in relation to patientbody weight. In particular aspects, the dosage may be about 0.1 mg/kg toabout 10 mg/kg. The dosages as indicated above may be administered onceper day, twice per day, three times per day, or more, as needed.Administration may be made with food, or before a meal. The appropriatedosage and dosage form will be readily determined by a person of skillin the art.

Various routes of administration may be used for the compositions,including enteral administration and oral administration. Oraladministration may be by tablet, capsule, sachet, drops, elixir,linctus, solution, emulsion, suspension, draught, puree, paste, syrup,gel, jelly, tonic, or other known means. Enteral administration may beby duodenal tubing or gastric tubing, including nasogastric tubing.Different means of administration are known in the art and may beutilised by a skilled person. The compositions disclosed herein are notlimited to a particular form for administration.

It may be useful to add one or more phenolic compounds to thecompositions of the invention, to further supplement the phenolicactivity therein. Exemplary compounds include but are not limited to:phenolic derivatives such as phenolic acid, and flavonoids such aslignins, proanthocyanidins, anthocyanins, anthocyanidins, isoflavones,catechins, tannins, quercetin, naringenin, and hesperidin. Specificanthocyanin compounds of interest are described herein. Particularlyencompassed are phenolic compounds extracted from one or more of: tea,cocoa, wine, soybeans, feijoa, citrus fruits, apples, pears, grapes,berries, and kiwifruit. Specific phenolics include but are not limitedto: ellagic acid, chlorogenic acid, catechin, epicatechin, kaemferol,E-caffeoyl-3-glucoside, E-caffeoyl-4-glucoside, neochlorogenic acid,phlorizin, procyanidin B1 and B2, quercetin, quercetin rhamnoside, andquercetin rutinoside.

As additional aspects, the compositions of the invention may beco-administered with one or more respiratory aids. A respiratory aid maybe a medication, prescription or non-prescription, or an alternativetreatment, such as a herbal remedy, or an essential oil, e.g., forvaporisation and/or inhalation. Of particular interest is use of thecomposition of the invention as a respiratory treatment during and/orfollowing other respiratory treatments. For example, the composition maybe formulated as a combined dosage form with one or more medicines oralternative treatments. Alternatively, the Boysenberry composition maybe administered as a separate dosage form along with one or moremedications or alternative treatments. A respiratory aid may have one ormore physiological effects, for example, anti-inflammatory,anti-spasmodic, bronchodilation, and/or muscle relaxation effects. Anyrespiratory aid may be long or short acting, and may be directed to aparticular disorder, such as asthma, chronic obstructive pulmonarydisease, etc.

Exemplary medications include but are not limited to bronchodilators,including short-acting bronchodilators such as albuterol (e.g., VospireER), levalbuterol (e.g., Xopenex), ipratropium (e.g., Atrovent),albuterol/ipratropium (e.g., Combivent), corticosteroids such asfluticasone (e.g., Flovent, Flovent Diskus, Flovent HFA), budesonide(e.g., Pulmicort, Pulmicort Flexhaler), mometasone (e.g., Asmanex),beclomethasone (e.g., QVAR), flunisolide (e.g., Aerospan), prednisolone,methylprednisolone, and hydrocortisone, methylxanthines such astheophylline (e.g., Theochron, Theo-24, Elixophyllin), long-actingbronchodilators such as aclidinium (e.g., Tudorza), arformoterol (e.g.,Brovana), formoterol (e.g., Foradil, Perforomist), glycopyrrolate (e.g.,Seebri Neohaler), indacaterol (e.g., Arcapta), olodaterol (e.g.,Striverdi Respimat), salmeterol (e.g., Serevent), tiotropium (e.g.,Spiriva), and umeclidinium (e.g., Incruse Ellipta), combinations of twoor more long-acting bronchodilators such as glycopyrrolate/formoterol(e.g., Bevespi Aerosphere), glycopyrrolate/indacaterol (e.g., UtibronNeohaler), tiotropium/olodaterol (e.g., Stiolto Respimat),umeclidinium/vilanterol (e.g., Anoro Ellipta).

Further exemplary medications include but are not limited tocombinations of inhaled corticosteroid(s) and long-actingbronchodilator(s) such as budesonide/formoterol (e.g., Symbicort),fluticasone/salmeterol (e.g., Advair, Advair Diskus), andfluticasone/vilanterol (e.g., Breo Ellipta), phosphodiesterase-4inhibitors such as roflumilast (e.g., Daliresp), beta agonists,including short-acting beta agonists such as albuterol (e.g., ProAirHFA, Ventolin HFA), and levalbuterol (e.g., Xopenex HFA),anticholinergics such as ipratropium bromide (e.g., Atrovent HFA),long-acting beta antagonists (LABAs) such as formoterol (Perforomist),and salmeterol (e.g., Serevent Diskus), leukotriene modifiers such asmontelukast (Singulair), zafirlukast (Accolate), and zileuton (e.g.,Zyflo, Zyflo CR), immunomodulators such as mepolizumab (Nucala),omalizumab (e.g., Xolair), reslizumab (e.g., Cinqair), bronchodilatorssuch as epinephrine (e.g., Primatene Mist, Bronkaid, Asthmahaler),ephedrine, and theophylline-ephedrine (e.g., Primatene tablets).

EXAMPLES

The examples described herein are provided for the purpose ofillustrating specific embodiments of the invention and are not intendedto limit the invention in any way.

Example 1: OVA-Induced Chronic Airway Inflammation and Oral Treatmentwith Boysenberry

Overview

Lung fibrosis negatively impacts on lung function in chronic asthma andis linked to the development of profibrotic macrophage phenotypes.Epidemiological studies have found that lung function benefits fromincreased consumption of fruit high in polyphenols. However, previousstudies have not investigated Boysenberry compositions, or effects onfibrotic or remodelling in airway systems.

The inventors investigated the effect of Boysenberry consumption, inboth therapeutic and prophylactic treatment strategies in a mouse modelof chronic antigen-induced airway inflammation. Boysenberry consumptionreduced collagen deposition and ameliorated tissue remodelling alongsidean increase in the presence of CD68+CD206+ arginase alternativelyactivated macrophages in the lung tissue. The decrease in tissueremodelling was associated with increased expression of profibrolyticmatrix metalloproteinase-9 protein in total lung tissue.

The inventors identified alternatively activated macrophages in the micethat consumed Boysenberry as a source of the matrix metalloproteinase-9.The inventors hypothesise that oral Boysenberry treatment moderatechronic tissue remodelling by supporting the development ofprofibrolytic alternatively activated macrophages expressing matrixmetalloproteinase-9. Regular Boysenberry consumption therefore has theability to moderate chronic lung remodelling and fibrosis in asthma andother chronic pulmonary diseases.

Materials

Anti-actin (clone AC-15), ovalbumin (OVA), 4% formalin, Tween 20,trans-hydroxyproline, 3,3′-diaminobenzidine (DAB) substrate,ketamine/xylazine, and all other chemicals were obtained from Sigma(Auckland, NZ). Alum was obtained from Serya (Heidelberg, Germany). TheBoysenberry juice was obtained as New Zealand 65 Brix Boysenberry juiceconcentrate kindly provided by Berryfruit Export NZ, currently tradingas Boysenberries New Zealand Ltd (Nelson, New Zealand). The 65 BrixBoysenberry juice concentrate from Berryfruit Export NZ was diluted insterile water to obtain a concentrate of 100 mg/ml total anthocyanins.From this, a further dilution was prepared to obtain a dosage of 10mg/kg of total anthocyanins. This further dilution is noted asBoysenberry solution.

Anti-mouse polyclonal inducible nitric oxide synthase (iNOS) (ab3523),arginase, TIMP-1 (ab38978), and MMP-9 (ab38898) were obtained from Abcam(Cambridge, UK). Antibodies against mouse CD68 (clone FA-11) CD3e, CD8a,CD4, CD11b, CD11c, and Gr-1 were obtained from BioLegend (San Diego,Calif.) and anti-CD206 (clone MR5D3) was obtained from AbDSerotec(Oxford, UK). Anti-mouse SiglecF, MHCII, and CD45 were from BDBiosciences (San Jose, Calif.).

TGFβ ELISA kit was obtained from R&D Systems (Minneapolis, Minn.).Vectastain Elite ABC staining kit was from Vector Laboratories(Burlingame, Calif.). Bio-Plex multiplex cytokine assays for IL-4, IL-5,IL-6, IL-13, and IFNγ, DC Lowry protein assay kit, and PVDF membranewere from Bio-Rad (Hercules, Calif.). BSA, NuPage 4-12% gels, MESrunning buffer, sample loading buffer, Novex sharp prestained, andMagicMark XP protein standards and all other buffers were from LifeTechnologies (Auckland, NZ).

Animals

C57BL/6J male mice were bred and group housed (5 per cage) inconventional polycarbonate cages with a filter top, in a specificpathogen-free animal facility at the Malaghan Institute of MedicalResearch, Wellington, New Zealand. All experimental procedures wereapproved by the Victoria University of Wellington Animal EthicsCommittee (approval number 2011R3M).

Mice were maintained on a 12-h light-dark cycle, at 21±2° C. ambienttemperature with freely available irradiated standard laboratory rodentchow (Specialty Feeds, Glen Forrest, Wash., Australia) and acidifiedwater.

OVA-Induced Chronic Airway Inflammation and Oral Boysenberry Treatment

Six-week-old mice were randomized into experimental groups (n=10 pergroup) and primed intraperitoneally (i.p.) with 100 μg OVA in 200 μlalum adjuvant on day 0. On day +7 mice were challenged intranasally(i.n.) with 100 μg OVA or PBS.

To establish chronic disease the i.n. challenge was repeated weekly(FIGS. 1A and 6A). Four days following the last i.n. OVA challenge micewere euthanized (ketamine/xylazine overdose) and bronchial-alveolarlavage fluid (BALF), serum, mediastinal lymph nodes and lung tissue werecollected.

For the treatment studies mice were fasted overnight before being orallygavaged with 250 μl of Boysenberry solution (see above); dosage at 10mg/kg of total anthocyanins) or sterile water on the day of OVAchallenge and again 2 days post-OVA challenge (FIGS. 1A and 6A).

Clodronate Liposome Depletion of Lung Macrophages and Tissue Analysis

Clodronate liposomes were prepared as previously described (58). ChronicOVA-induced tissue damage was established over 5 weeks. Mice were thentreated intranasally with 100 μl clodronate liposomes the day prior toeach oral gavage with 250 μl of Boysenberry solution (see above; dosageat 10 mg/kg of total anthocyanins) or sterile water (FIG. 6A). Two daysfollowing the last oral gavage mice were euthanized (ketamine/xylazineoverdose) and BALF, serum, mediastinal lymph nodes, and lung tissue werecollected.

Cells isolated from the BALF were stained for key surface markers toidentify monocytes/macrophages (CD45+/CD11b+/Cd11c+/MHCIIlow) andeosinophils (CD45+/CD11b+/siglecF+) by flow cytometry as previouslydescribed (52). TGFβ ELISA and Bio-Plex multiplex cytokine assays wereperformed on lung tissue supernatants following the manufacturer'sinstructions. Lung tissue was fixed in 4% formalin, sectioned, andstained with hematoxylin and eosin (H&E), Masson's Trichrome or Alcianblue-periodic acid-Schiff (AB-PAS) stains (Dept. of Pathology,Wellington School of Medicine, University of Otago, Wellington, NZ).

Further sections were cut for immunological labelling. Lung sectionswere incubated with biotin-conjugated MMP-9, then labelled with DAB andcounter-stained with hematoxylin. Other tissue sections were incubatedwith fluorescently labelled CD68 (31), CD206 (57), and arginase or MMP-9(44), then counterlabelled with DAPI-containing mounting medium.

All sections were imaged on an Olympus BX51 compound microscope andcaptured by using cellSens (Olympus NZ) software, bright light in colourand fluorescence in grayscale. Fluorescence images were processed(cropped, false coloured, and merged) in Pixelmator image software(Vilnius, Lithuania). Fluorescently labelled cells were quantified byfour independent, blinded observers. Cells were counted in random fieldsfrom multiple animals and scored as negative, single positive, or doublepositive for CD68, CD206, arginase, or MMP-9. Data were expressed as apercentage of total cells counted.

Biochemical and Molecular Biological Tissue Analysis and StatisticalAnalysis

Biochemical and molecular biological tissue analysis. Lung tissue wassnap frozen and stored at −70° C. Lung collagen was quantified by thehydroxyproline assay as previously described (2). For Western blotting,tissue was homogenized in protein lysis buffer (Tris-HCl, NaCl, 10%Nonidet P-40, 10% sodium deoxycholate, 100 nM EDTA, pH 7.4 with proteaseand phosphatase inhibitors). Protein concentration was quantified by aLowry protein assay as per the manufacturer's instruction.

Samples (30 μg protein) were separated by SDS-PAGE gel electrophoresisunder reducing conditions and transferred onto PVDF membrane.Nonspecific protein binding was blocked with 3% BSA (10 mM PBS with 0.2%Tween 20) and the membranes were probed overnight with primaryantibodies specific to iNOS (64), arginase (53), MMP-9 (44), and TIMP-1(55), or β-actin (12) loading control (4° C.). Membranes were washed andincubated with horseradish peroxidase-conjugated secondary antibodiesand visualized by chemiluminescence on a Carestream Gel Logic Pro 6000imager. Protein expression was densitometrically quantified andnormalized to β-actin with ImageJ's Gel analysis tool (50). Images wereprocessed and cropped in Pixelmator image software.

Data were analysed by one-tailed Student's t-test for comparisonsbetween two groups or one-way ANOVA with Tukey's post hoc test forcomparisons between three or more groups as indicated (Prism, GraphPad,San Diego, Calif.). P<0.05 or less was considered statisticallysignificant.

Results—Boysenberry Consumption Ameliorates OVA-Induced Chronic AirwayInflammation

To investigate the effect of Boysenberry treatment on established lungremodelling, mice were challenged weekly with intranasal OVA for 5weeks, then challenged weekly with OVA for an additional 5 weeksalongside weekly oral treatment with Boysenberry (FIG. 1A).

As shown in FIG. 1B, lung tissue from OVA-challenged mice exhibitedincreased cellular infiltrate and loss of lung structure. OVA-inducedcellular infiltrate and lung damage were decreased inBoysenberry-treated mice (FIG. 1B). Staining of lung tissue for mucusproduction identified fewer mucus-positive cells in OVA-challenged micereceiving Boysenberry treatment compared with OVA only-challenged mice(FIG. 1C). Boysenberry treatment alone had no effect on cellularinfiltration, lung structure, or mucus production.

Results—Boysenberry Treatment Increases AAMs in the Lung ofOVA-Challenged Mice

H&E-stained lung tissue sections showed more macrophages inOVA/Boysenberry-treated mice compared with OVA mice (FIG. 2A).Immunoblot analysis of lung tissue identified a decrease in iNOSexpression in the lung tissue of OVA/Boysenberry-treated mice comparedwith OVA challenge alone (FIGS. 2 , B and C). At the same time, anincrease was observed in arginase expression in OVA-challenged mice(FIGS. 2 , B and D). that was further enhanced in OVA-challenged micetreated with Boysenberry. Arginase expression was not affected byBoysenberry treatment alone.

AAMs expressing arginase are closely associated with lung remodelling(29). To determine whether the observed lung macrophages werealternatively activated, lung tissue was stained with fluorescentlylabelled antibodies for the macrophage marker CD68 and the AAM markersCD20 and arginase.

Lung tissue from OVA/Boysenberry-treated mice showed an increase inCD68+CD206+arginase+ macrophages compared with OVA-challenged mice (FIG.3 ). Quantitative analysis of the CD68+CD206+arginase+ macrophagesfurther confirmed a significant increase in the percentage ofCD68+CD206+arginase+ macrophages in the lung tissue ofOVA/Boysenberry-treated mice compared with OVA-challenged mice(60.00±3.54% compared with 23.47±5.61%, P<0.001, one-tailed Student'st-test). Together these data identify an increase in the number of lungmacrophages expressing an alternatively activated phenotype inOVA-challenged mice receiving Boysenberry treatment.

Results—Boysenberry Treatment Decreases OVA-Induced Collagen Depositionand Increases MMP-9 Expression in the Lung

Increased AAMs and arginase expression are commonly associated withtissue fibrosis (14, 27, 66); therefore the effect of Boysenberrytreatment was investigated for OVA-induced collagen deposition in thelung. Following this the levels of hydroxyproline were measured in thelung tissue as a surrogate marker of collagen deposition (2, 63).

OVA challenge alone resulted in abnormal collagen deposition in theairways with signs of collagen invasion throughout the lung tissue thatwas abrogated in the lungs of OVA/Boysenberry-treated mice (FIG. 4 ). Inaddition, there was a significant drop in the levels of hydroxyprolinein the lungs of OVA-challenged mice treated with Boysenberry, confirmingthat Boysenberry treatment ameliorated OVA-induced collagen deposition(FIG. 4B). Boysenberry restored the OVA-induced decrease in the levelsof TGFβ in the lung (FIG. 4C) but did not affect the levels of IL-4,IL-5, IL-6, IL-13, or IFNγ (data not shown).

To determine how Boysenberry treatment could be moderating lung fibrosisthe expression of MMP-9 was measured in the lung tissue by immunoblot.

It was determined that MMP-9 expression was increased in OVA-challengedmice treated with Boysenberry compared with mice challenged with OVAalone (FIG. 4D). Boysenberry treatment alone did not affect MMP-9 levelsin the lung. Tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) isthe endogenous inhibitor of MMP-9 (49). The ratio of TIMP-1/MMP-9expression significantly increased in the lung tissue of chronicOVA-challenged mice and this increase was reversed with Boysenberrytreatment (FIG. 4E). These results indicate that Boysenberry-mediatedreduction in collagen deposition and tissue remodelling was associatedwith elevated production of fibrolytic MMP-9 and a subsequent rebalancein the ratio of TIMP-1/MMP-9.

Results—Alternatively Activated Macrophages are a Source of MMP-9Protein in the Lungs of OVA/Boysenberry-Treated Mice

Lung tissue slides were analysed to identify potential cellular sourcesof MMP-9. DAB-MMP-9 staining identified a high degree of MMP+ cellsexhibiting macrophage morphology in OVA/Boysenberry-treated micecompared with OVA-treated controls (FIG. 5A). Immunofluorescent staining(FIG. 5B) and quantitative analysis of the lung tissue confirmed thatthere were more MMP-9+/CD206+/CD68+ cells present inOVA/Boysenberry-treated lungs than those challenged with OVA alone(39.30±6.39 vs. 21.07±5.82%; P<0.05, one-tailed Student's t-test). Theseresults identify CD206+/CD68+ AAMs as a source of the increased MMP-9protein levels.

Results—Depletion of Lung Macrophages Reduces the Beneficial Effect ofBoysenberry Consumption on Tissue Remodelling in Established ChronicLung Inflammation

Next, the inventors looked at the effect of depleting lung macrophageson the beneficial effects of Boysenberry on chronic lung inflammation.Macrophages were depleted by administration of clodronate liposomesafter establishing chronic lung inflammation and remodelling, and priorto administration of each Boysenberry treatment (FIG. 6A). It wasconfirmed that significant depletion of the lung macrophages had beenobtained by flow cytometry (FIG. 6B) and that this was associated with asignificant reduction in hydroxyproline levels in the lung ofOVA-challenged mice treated with Boysenberry (FIG. 6C). These dataindicate that Boysenberry requires macrophages to mediate its beneficialeffects on lung tissue remodelling.

Results—Boysenberry Treatment Prophylactically Prevents OVA-InducedAirway Inflammation

Finally, the effect of Boysenberry treatment was tested using aprophylactic dosing regimen (FIG. 7A). Again, Boysenberry treatmentresulted in abrogation of OVA-induced tissue remodelling andsignificantly reduced cells in the lung lavage fluid (FIG. 7 , B-D),This was associated with lower levels of hydroxyproline in the lungtissue and a decrease in the ratio of TIMP-1/MMP-9 expression (FIG. 7 ,E-G).

Discussion

Fruit consumption has been linked with improved lung function in asthmasufferers and the amelioration of acute airway inflammation inexperimental models (16, 19, 40). However, no findings have beenestablished in these studies in relation to Boysenberry compositions,airway fibrosis, or airway remodelling, and it is well established thatother known asthma treatments have failed to address airway remodelling.

It is demonstrated herein that consumption of a Boysenberry compositionmoderates chronic lung remodelling and fibrosis in both a therapeuticand a prophylactic setting. Furthermore, the data indicate thatmacrophages play an important role in Boysenberry-mediated protectionand that this protection may result from modulation of AAMs andincreased MMP-9 activity.

An increase in both arginase activity (26, 27, 41) and AAMs (4, 9) isoften linked with asthma pathogenesis. However, there is evidence thatthe presence of AAMs does not specifically underpin the development ofallergic asthma (37), which indicates that AAMs may play an alternativerole.

As shown herein, the Boysenberry treatment increased the population ofarginase-positive AAMs alongside a drop in iNOS expression in the lungtissue of chronic OVA-challenged mice. Arginase and iNOS play aninteractive role in regulating lung inflammation and repair (34, 35,66). Where iNOS activity is associated with active inflammation,arginase expression is indicative of a switch toward inflammatoryresolution (35, 63). Boysenberry consumption therefore appears torebalance the lung environment, supporting inflammation resolution bymodulating the functional phenotype of AAMs in the lung.

The presence of AAMs has been associated with decreased Th-2 cytokineproduction in lung inflammation (36, 42). However, it was determinedthat no changes in the levels of Th-2 cytokines IL-4, IL-5, and IL-13with Boysenberry consumption following OVA challenge. This indicatedthat inhibition of proinflammatory Th-2 cytokine production by AAMs wasnot contributing to the protective effect of Boysenberry treatment.

Clinical and animal data indicate that the role of MMP-9 in asthma ismultifaceted. Lung macrophages producing MMP-9 have been identified inboth experimental and clinical settings (1, 5, 49). Elevated levels ofactive MMP-9 have been found in plasma and sputum samples from patientswith asthma, compared with healthy controls (3, 23). Increased MMP-9expression has been correlated with acute asthma exacerbation, includingincreased lung eosinophilia (6, 23). Conversely, an increase in MMP-9levels has been associated with improved lung function in airway disease(25, 65). MMP-9 overexpression has also been shown to have beneficialeffects in a model of pulmonary fibrosis (5). In contrast, data fromMMP-9 knockout mice show a partial reduction in the development ofasthma symptoms and reduced remodelling but, in some cases, a lack ofMMP-9 has been shown to exacerbate disease (15, 24, 32).

MMP-9 exerts many downstream effects on different immune parameters,including the activation of both pro- and anti-inflammatory cytokines(15). Nevertheless, the data shown herein indicate thatBoysenberry-induced protection of lung tissue from chronic collagendeposition and fibrosis is orchestrated, in part, through the generationof fibrolytic AAM producing MMP-9. Consistent with this, the data showthat depletion of macrophages during the resolution phase ofinflammation leads to increased collagen deposition with Boysenberryconsumption. A similar resolution-promoting role for macrophages hasbeen illustrated in bleomycin-induced pulmonary fibrosis (14).

Matrix metalloproteinases are regulated by their natural inhibitorsTIMPs, and high TIMP-1/MMP-9 ratios are proposed to favour collagendeposition and lung remodelling (21, 28, 38). Here a significantincrease was observed in the ratio of expression of TIMP-1/MMP-9 in thelung tissue of chronic OVA-challenged mice and this was reversed byBoysenberry treatment. The drop in the ratio of TIMP-1/MMP-9 inBoysenberry-treated mice therefore represents a potentially beneficialre-adjustment in the regulation of collagen deposition and breakdown.

TGFβ is associated with both normal (20) and pathological (17, 22, 56)tissue repair processes through its role in extracellular matrixproduction. In this study, it was observed that chronic OVA challengeled to a decrease in TGFβ levels that was reversed by Boysenberryconsumption. There is evidence that TGFβ lowers the TIMP-1/MMP-9 ratio,thus favouring a more fibrolytic environment (18, 54, 56). As such theincrease in TGFβ levels observed in the lungs of OVA-challenged micefollowing Boysenberry treatment could serve to limit excessive tissuefibrosis and inappropriate remodelling during lung repair by loweringthe TIMP-1/MMP-9 ratio. TGFβ is also known to stimulate fibroblastcontraction for normal tissue repair (20), which could likewisecontribute toward the beneficial effects of Boysenberry treatment. Assuch the elevation of TGFβ has the potential to promote ananti-inflammatory, pro-resolution environment within the lung viamultiple mechanisms.

The results from these studies show that Boysenberry administrationexhibits a beneficial effect on chronic lung fibrosis in both atherapeutic and a prophylactic setting. This indicates that Boysenberryconsumption may help avoid inappropriate fibrotic remodelling in casesof both poorly controlled and well-controlled asthma. Finally, thesefindings provide the first evidence that Boysenberry consumption couldbe used to support the development of fibrolytic AAMs with the potentialto regulate appropriate lung remodelling in asthma and other lungconditions exhibiting fibrotic pathologies.

In summary, these findings have showed that Boysenberry compositions maybe used to decrease inflammation and aberrant collagen deposition in therespiratory tract, and thereby find use in the treatment and preventionof various disorders of the airways, including asthma, chronicobstructive pulmonary disease, reactive airway disease, airway fibrosis,and airway remodelling.

Example 2: OVA-Induced Acute Airway Inflammation and Oral Treatment withBoysenberry and Apple

Materials and Methodology

Six-week-old mice treated and assessed as in Example 1, noted above withthe following modifications. The tested solutions included: Boysenberry1 and Boysenberry 10, 0.67% or 6.7%, respectively, apple 1 and apple 1010, 1.87% and 18.7%, respectively, BerriQi™ Boysenberry with apple 1 andBerriQi™ Boysenberry with apple 10, 0.67%/1.87% and 6.7%/18.7%,respectively. Commercial Boysenberry juice concentrate was obtained asdescribed in Example 1. Apple juice concentrate was supplied fromInfruit Ltd (Titirangi, Auckland, New Zealand), as manufactured byProfruit (2006) Ltd (Hastings, New Zealand).

For the 18.7% solutions, 18.7 g juice concentrate was diluted in 100 gwater. For the 6.7% solutions, 6.7 g juice concentrate was diluted with100 g water. For the 1.87% solutions, 1.87 g juice concentrate wasdiluted in 100 g water. For the 0.67% solutions, 0.67 g juiceconcentrate was diluted in 100 g water. For the combined 6.7%/18.7%solutions, 6.7 g and 18.7 g for the respective juice concentrate wasdiluted in 100 g water. For the combined 0.67%/1.87% solutions, 0.67 gand 1.87 g for the respective juice concentrate was diluted in 100 gwater. In reference to the combined solutions, the Boysenberry to applepercentage was 27% to 73%. Prepared in parallel, BerriQi™ Boysenberrywith apple test solutions were heated for 8 hours at 80° C. prior toadministration to test for deactivation of anti-inflammatory activity.

The protocol for testing the combined administration of Boysenberry withapple administration was based on previously published methods forinducing allergic airways inflammation (52). The protocol for acuteinflammation utilised an 11 day model. To evaluate the efficacy ofBoysenberry, apple, combined Boysenberry and apple administration, anddetermine the effect of diluting the treatments 10-fold the followingtreatment groups were tested: 1) Baseline control (no disease); 2)Disease control; 3) Apple 10 (at 18.7%)+disease; 4) Apple 1 (at1.87%)+disease; 5) Boysenberry 10 (at 6.7%)+disease; 6) Boysenberry 1(at 0.67%)+disease; 7) BerriQi™ Boysenberry with apple 10 (at6.7/18.7%)+disease; 8) BerriQi™ Boysenberry with apple 1 (at0.67/1.87%)+Disease; 9) ‘Cooked’ BerriQi™ Boysenberry with apple (at6.7/18.7%)+disease. Each group included 10 animals. Experiments wererepeated three times. Statistical analysis was performed as described inExample 1.

As per prior employment of this model, the following dosing/challengeregimen was used. Mice were primed for allergic airways inflammation 7days prior to the challenge and samples were collected 4 days postchallenge. Mice were administered (treated) with 250 μl of the notedtreatments or water (control) on day 0 and day+2 (see below). For theBoysenberry 10 test solutions (Boysenberry 10 and BerriQi™ Boysenberrywith apple 10), the dosage was administered to deliver 4.87 mg/kg totalanthocyanins. For the Boysenberry 1 test solutions (Boysenberry 1 andBerriQi™ Boysenberry with apple 1), the dosage was administered todeliver 0.487 mg/kg total anthocyanins.

The following samples were collected: 1) lung wash; 2) lung tissue; 3)mediastinal lymph node; 4) blood. These samples were analysed for thefollowing as per the methodologies outlined in (52): 1) total cellcounts (and cellular composition): lung wash, lung tissue, and lymphnode; 2) cytokine and chemokine production including IL-4, IL-5, IL-6,IL-10, IL-13, CCL11, IFNγ: lung wash, lung tissue and blood; 3) IgE,IgG1: blood only. Cytokine levels were tested using BioLegendLEGENDplex™ multi-analyte flow assay kit in accordance with themanufacturer's instructions.

Results

FIG. 8 shows the testing schematic. From the results obtained, ovalbuminchallenge increased the appearance of inflammatory cells within thelung. This increase was reduced by particular treatments back to naïvelevels, i.e., without OVA addition.

The results demonstrated increased total immune cell infiltration intothe lung in mice challenged intranasally with the allergen, OVA (FIG. 9). Treatment with apple alone did not reduce the cellular infiltration.BerriQi™ Boysenberry with apple reduced the cellular infiltration atboth concentrations tested, and this was reversed when the BerriQi™Boysenberry with apple was heated to 80° C. for 8 hours.

The results showed that apple treatment alone had little effect oneosinophil numbers, neutrophils, monocytes, or antigen presenting cells(APCs; FIGS. 10, 14-16 ). In contrast to this, combined BerriQi™Boysenberry with apple treatment substantially reduced the number ofeosinophils, neutrophils, and monocytes at low dosage levels (FIGS. 10,14, 15 ). Heating the BerriQi™ Boysenberry with apple solution prior totreatment reversed this effect (FIGS. 10, 14, 15 ). Combined BerriQi™Boysenberry with apple treatment also reduced the number of APCs (FIG.16 ). Heating reversed this effect (FIG. 16 ).

Haematoxylin and eosin staining showed that the ovalbumin challengeresulted in tissue swelling and immune cell infiltration, while combinedBerriQi™ Boysenberry with apple treatment appeared to reduce tissueswelling compared to OVA alone (FIG. 13 ).

AB-PAS staining showed mucous production was variable between thedifferent treatments (FIGS. 17-19 ). None of the treatments made mucousproduction worse, although there were more mucous-producing cellsobserved in the lowest fruit concentrations, and none of the treatmentsappeared to prevent increased mucous production (FIGS. 17-19 ). Theseresults were attributed to the short duration and small number ofadministrations (two) for the acute experimental model. This contrastedto the longer experimental testing period and additional administrationsnoted in Example 1, noted above. It was proposed that effects on mucusproduction could be better observed given longer testing times andadditional dosages.

Masson's trichrome staining showed that acute OVA challenge did notsubstantially increase fibrosis. There was not an increase in collagendeposition within the lung tissue (FIGS. 20-22 ). The results from thishistology indicated that the effect of treatments on fibrosis could notbe tested in this acute experimental model. This confirmed that theshort duration of the acute experimental model did not providesufficient time to see effects for longer-term symptoms such as mucusproduction and collagen deposition.

Lung fluid obtained from test and control animals was assessed forcytokine levels, including granulocyte-macrophage colony-stimulatingfactor (GM-CSF), IFNγ, IFNβ, IL-1α, IL-1β, TNFα, IL-12(p40), IL-10,IL-6, IL-27, CXCL1, and CCL2. Boysenberry treatment alone reduced GM-CSFlevels (FIG. 23 ), while apple treatment and combined BerriQi™Boysenberry with apple treatment did not reduce GM-CSF levels (FIG. 23). CCL11 levels did not appear to be significantly affected (FIG. 24 ).IFNγ, IFNβ, IL-1α, IL-1β, TNFα, IL-12(p40), IL-10, IL-6, IL-27, CXCL1,and CCL2 were not detected in the samples tested (data not shown). Basedon this, it was postulated that the effects of the BerriQi™ Boysenberrywith apple treatment were not mediated by CCL11 secretion.

Discussion

From the results, it was concluded that combined BerriQi™ Boysenberrywith apple treatment reduced the number of infiltrating eosinophils atboth the standard and lower dosage levels. This effect was dependent ontemperature-sensitive elements of the BerriQi™ Boysenberry with applecomposition. Treatment using apple alone had little effect on numbers ofneutrophils, monocytes, or APCs. Combined BerriQi™ Boysenberry withapple treatment reduced the number of neutrophils and monocytes at lowerdosage levels, and reduced the number of APCs at standard and lowerdosage levels. These effects were dependent on temperature-sensitiveelements. It is clear, then, that combined administration of Boysenberryand apple compositions can be used to reduce numbers of immune cellsassociated with allergic airways inflammation.

The assessment of mucous production was variable between the differenttreatments, leading to the conclusion that additional or alternativemeasures of mucous suppression such as IL-13, IL-9 production mayprovide further clarity. Analysis of lung tissue supernatant to identifychanges in proinflammatory cytokines showed that CCL11 levels wereunaffected by BerriQi™ Boysenberry with apple treatment, suggesting thatthe decrease in eosinophils was not mediated via inhibition of CCL11.Additional analysis may be used to identify the particular agents thatare involved in this process.

Overall, the results of this acute study were positive, showing thatBerriQi™ Boysenberry with apple treatment reduced the cellularinfiltration at both concentrations tested, whereas apple alone did notat either concentrations tested. Boysenberry alone also reduced cellularinfiltration which was similar to what was found in the previousresearch described herein (see, e.g., Example 1 and (74)). Notably, theacute model of allergic-airways inflammation used in this study did notsufficiently promote the development of tissue fibrosis, so it could notbe determined if BerriQi™ Boysenberry with apple treatment was able topromote the development of anti-fibrosis macrophages and prevent tissuedamage. To determine the presence of such effects, it has been necessaryto carry out further studies on chronic allergic-airways inflammation.

Example 3: OVA-Induced Chronic Airways Inflammation and Oral Treatmentwith Boysenberry and Apple

Overview

While consumption of certain fruits and vegetables has been studied inrelation to beneficial health effects (87, 88, 91-94), the experimentsdescribed herein have been the first to show a substantial beneficialeffect for specific Boysenberry compositions. See Example 1 and 2, andalso (74). The key findings from the studies of Example 1 include: 1)Boysenberry consumption significantly reduced allergen-induced airwaysinflammation through decreased cell infiltration and increasedanti-inflammatory protein production; 2) Boysenberry reduced collagendeposition and assisted in the repair of damaged tissue repair bysupporting the development of fibrolytic macrophages, a type of immunecell; and 3) Boysenberry treatment prophylactically prevents ovalbumin(OVA)-induced airways inflammation.

The effect of different apple varieties on key cytokines for allergicairways disease has also been investigated (96), and cytokine inhibitoryability has been established for apple varieties in a cell culture modelof allergic asthma induction. The inhibitory ability was correlated tothe presence of the procyanidin polyphenols (96), and it has been shownthat these compounds, in isolation, are potent inhibitors of keyallergic chemokines CCL11 (90) and CCL26 (89). The aim of the currentproject was to evaluate BerriQi™ Boysenberry with apple treatment, anovel combination of Boysenberry and apple juice concentrates and water,in an animal model of chronic OVA-induced allergic airways inflammation.

Materials and Methodology

To perform this study, the previously established mouse model and anoral dosing strategy of chronic OVA-induced allergic airwaysinflammation was utilised as in Example 1 (see also, (52), (97)).Briefly, mice were primed with OVA/Alum intraperitoneally (i.p.) andthen challenged 7 days later with OVA intranasally (i.n.). These i.n.challenges were performed every week for 10 weeks. After 5 weeks of OVAchallenges, the BerriQi™ interventions were begun. For theseinterventions, mice were fasted for 4 hours before being orally gavagedwith water (disease and vehicle control), BerriQi™ Boysenberry withapple at 100%, 50% or 25%, at 2 days prior, at 1 hour prior to i.n. OVAchallenge, and at 2 days post OVA challenge.

The intervention groups were: 1) Naïve (baseline control); 2) +OVA(disease and water vehicle control); 3) +OVA+BerriQi™ 100% (disease plus100% BerriQi™ Boysenberry with apple) containing New Zealand sourced 70°Brix apple juice concentrate and New Zealand sourced 65° BrixBoysenberry juice concentrate; 4) +OVA+BerriQi™ 50% (disease plus 50%BerriQi™ Boysenberry with apple) containing New Zealand sourced 70° Brixapple juice concentrate and New Zealand sourced 65° Brix Boysenberryjuice concentrate; and 5) +OVA+BerriQi™ 25% (disease plus 25% BerriQi™Boysenberry with apple) containing New Zealand sourced 70° Brix applejuice concentrate and New Zealand sourced 65° Brix Boysenberry juiceconcentrate. The Boysenberry juice concentrate was obtained fromBoysenberry NZ (Nelson, New Zealand). The apple juice concentrate wassupplied by RD2 International (Auckland, New Zealand), and manufacturedby Profruit (Hastings, New Zealand).

The concentration of Boysenberry in BerriQi™ Boysenberry with apple wascalculated to deliver 0.73 mg/kg Boysenberry anthocyanins per serve fora 70 kg human, this being equivalent to 10 mg/kg in mouse. This dose wasselected based on the previous studies (see Example 1 and (52)) thatdetermined that consumption of 10 mg/kg Boysenberry anthocyaninsresulted in reduced inflammation and tissue fibrosis in a mouse model ofchronic allergic airways inflammation. 100% BerriQi™ Boysenberry withapple provided 10 mg/kg total Boysenberry anthocyanins for a 25 g mouse;50% and 25% BerriQi™ Boysenberry with apple provided 5 mg/kg and 2.5mg/kg total Boysenberry anthocyanins for a 25 g mouse, respectively.Details for the dosages are provided in Example 5, below.

Mice were euthanised 4 days following i.n. OVA challenge. The followingparameters were measured as described in Example 1: 1) cellularinfiltration: eosinophils, neutrophils, monocytes, and antigenpresenting cells (APCs); 2) histological changes: haematoxylin and eosin(H&E), Alcian blue and periodic acid-Schiff (AB-PAS), Masson's trichromestaining; and 3) collagen production using the hydroxyproline assay. Forthe hydroxyproline assay, the commercial colorimetric kit was used(AbCam ab222941).

Results

The results showed an increased total immune cell infiltration into thelung in mice challenged i.n. with the allergen, OVA (FIG. 25 ). BerriQi™Boysenberry with apple treatment significantly reduced the cellularinfiltration at the 50% and 25% concentrations tested (FIG. 25 ). The100% concentration BerriQi™ Boysenberry with apple treatment appeared tohave no effect on the OVA-induced increase in immune cells in the lung.This was postulated as showing a therapeutic window for the BerriQi™Boysenberry with apple treatment.

The immune cells were identified as eosinophils (FIG. 26 ), antigenpresenting cells (FIG. 27 ), and monocytes (FIG. 28 ). The number ofeosinophils showed a significant increase in mice challenged with OVA(FIG. 26 ). A decrease in eosinophil number was obtained by the 50%BerriQi™ Boysenberry and apple treatment (FIG. 26 ). Antigen presentingcells showed a significant increase with the OVA challenge, and adecrease in APC number was obtained by the 50% and the 25% BerriQi™Boysenberry and apple treatment (FIG. 27 ). The number of monocytes wasincreased in mice challenged with OVA, and a decrease in monocyte numberwas obtained by the 50% and the 25% BerriQi™ Boysenberry and appletreatment (FIG. 28 ).

Haematoxylin and eosin staining showed that the ovalbumin challengeresulted in tissue swelling and confirmed the immune cell infiltration,and that this was decreased by BerriQi™ Boysenberry and apple treatment(FIG. 29 ). AB-PAS staining showed that the ovalbumin challenge resultedin increased mucous production that was decreased in a dose-dependentmanner by BerriQi™ Boysenberry and apple treatment (FIG. 30 ).

Masson's trichrome staining showed that repeated OVA challenges resultedin diffuse blue staining of collagen fibres within the airways (FIG. 31). BerriQi™ Boysenberry and apple treatment reduced the appearance ofthese blue collagen fibres within the lung (FIG. 31 ).

Quantification of collagen levels with the hydroxyproline assay showedno increase in collagen with OVA challenges (FIG. 32 ), which wascontrary to previous results. No significant changes in collagen levelswere seen with 100% or 50% BerriQi™ Boysenberry and apple treatment(FIG. 32 ). However, a significant increase in collagen levels was seenwith 25% BerriQi™ Boysenberry and apple treatment (FIG. 32 ). This isdiscussed in more detail, below.

Discussion

The i.n. OVA challenge resulted in the appearance of increasedinflammatory cells within the lung, which was reduced by 50% and 25%BerriQi™ Boysenberry with apple treatment. The 100% BerriQi™ Boysenberrywith apple treatment had no effect on the number of infiltrating immunecells compared to the OVA challenged mice, indicating that there may bean optimal concentration for this treatment.

Mucous production was reduced by BerriQi™ Boysenberry with appletreatment in a dose-dependent manner, with the 25% BerriQi™ Boysenberrywith apple treatment mediating the greatest reduction in mucousproduction.

Analysis of lung tissue to quantify the changes in total collagen showedthat the quantity of collagen following OVA challenges was unchanged,which is inconsistent with the results from the original hydroxyprolineassay (see, e.g., Example 1 and (74)). The assay used in this Exampleemployed measurement reagents obtained from an alternate source ascompared to the original assay.

Notably, Masson's trichrome staining indicated that OVA challengesresulted in collagen infiltrating into the airways, and BerriQi™Boysenberry and apple treatment helped to reverse this infiltration.Thus, histological methods established that the location of the collagenaround the tissues is altered by OVA, and this can be addressed byBerriQi™ Boysenberry and apple treatment.

Further research is needed to fully elucidate the meaning of the presenthydroxyproline assay results. One possible explanation is the assaydifferences, as noted. In addition, it is possible that, although therewas no change in the quantity of total collagen from OVA challenge, thelocation of the collagen has been altered, and this is being addressedby the BerriQi™ Boysenberry with apple and blackcurrant treatment. It isalso possible that the increase in the amount collagen coupled with thereduction of collagen staining in the airways, as seen with the 25%BerriQi™ Boysenberry and apple treatment, is a result of tissueremodelling that occurs when the inflammation is being resolved.

Example 4: OVA-Induced Chronic Airways Inflammation and Oral Treatmentwith Boysenberry, Apple and Blackcurrant

Materials and Methodology

To perform this study, the mouse model and an oral dosing strategy ofchronic OVA-induced allergic airways inflammation was utilised as inExample 1. See also, (52), (97). Briefly, mice were primed with OVA/Alumintraperitoneally (i.p) and then challenged 7 days later with OVAintranasally (i.n), this i.n challenge every week for 10 weeks. After 5weeks of OVA challenges, the interventions were begun. For theinterventions, mice were fasted for 4 hours before being orally gavagedwith water (disease and vehicle control), or BerriQi™ Boysenberry withapple and blackcurrant at 100%, at 2 days prior, at 1 h prior to i.n.OVA challenge, and at 2 days post OVA challenge.

The intervention groups were: 1) Naïve (baseline control); 2) +OVA(disease and water vehicle control); 3) +OVA+BerriQi™ Boysenberry withapple and blackcurrant 100% (disease plus 100% BerriQi™ Boysenberry withapple and blackcurrant) containing New Zealand sourced 70 Brix applejuice concentrate, New Zealand sourced blackcurrant juice, and NewZealand sourced 65 Brix Boysenberry juice concentrate. The Boysenberryjuice concentrate was obtained from Boysenberry NZ (Nelson, NewZealand). The apple juice concentrate was supplied by RD2 International(Auckland, New Zealand), and manufactured by Profruit (Hastings, NewZealand). The blackcurrant juice concentrate was obtained from NewZealand Blackcurrant Co-operative Ltd (Nelson, New Zealand).

The concentration of Boysenberry in BerriQi™ Boysenberry with apple andblackcurrant oral composition was calculated to deliver 0.73 mg/kg totalanthocyanins per serve for a 70 kg human, this being equivalent to 10mg/kg in mouse. This dose was selected based on the previous studies(see Example 1 and (52)) that determined that consumption of 10 mg/kgBoysenberry anthocyanins resulted in reduced inflammation and tissuefibrosis in a mouse model of chronic allergic airways inflammation. The100% BerriQi™ Boysenberry with apple and blackcurrant oral compositionprovided 10 mg/kg total anthocyanins for a 25 g mouse. Details for thedosages are provided in Example 5, below.

Thus, in this study, it was tested whether a reduced concentration ofBoysenberry anthocyanins could be utilised by supplementing theBoysenberry juice concentrate with an equal amount of blackcurrant juiceconcentrate to make the total concentration of anthocyanins to 10 mg/kg.

Mice were euthanized 4 days following i.n. OVA challenge. The followingparameters were measured as described in Example 1: 1) cellularinfiltration: eosinophils, monocytes and antigen presenting cells(APCs); 2) histological changes: haematoxylin and eosin (H&E), Alcianblue and periodic acid-Schiff (AB-PAS), and Masson's trichrome staining;and 3) collagen production using the hydroxyproline assay. For thehydroxyproline assay, the commercial colorimetric kit was used (AbCamab222941).

Results

The results showed increased total immune cell infiltration into thelung in mice challenged i.n. with the allergen, OVA (FIG. 33 ). BerriQi™Boysenberry with apple and blackcurrant treatment significantly reducedthe cellular infiltration at the concentrations tested (FIG. 33 ).

The infiltrating cells were made up of eosinophils (FIG. 34 ), antigenpresenting cells (FIG. 35 ), and monocytes (FIG. 36 ). The number ofeosinophils was increased in mice challenged with OVA, but this increasewas not affected by BerriQi™ Boysenberry with apple and blackcurranttreatment (FIG. 34 ). The number of antigen presenting cells wassignificantly increased by OVA challenge, and the APC number wassignificantly decreased by BerriQi™ Boysenberry with apple andblackcurrant treatment (FIG. 35 ). Monocytes trended towards increasednumbers in mice challenged with OVA, and the monocyte numbers weredecreased by BerriQi™ Boysenberry with apple and blackcurrant treatment(FIG. 36 ).

Haematoxylin and eosin staining showed that the ovalbumin challengeresulted in tissue swelling and immune cell infiltration, which wasdecreased by BerriQi™ Boysenberry with apple and blackcurrant treatment(FIG. 37 ). AB-PAS staining showed that the ovalbumin challenge resultedin increased mucous production that was unaffected by BerriQi™Boysenberry with apple and blackcurrant treatment (FIG. 38 ).

Masson's trichrome staining showed that repeated OVA challenges resultedin diffuse blue staining of collagen fibres within the airways (FIG. 39). BerriQi™ Boysenberry with apple and blackcurrant treatment reducedthe appearance of these blue collagen fibres within the lung (FIG. 39 ).

The hydroxyproline assay showed that there was no increase in totalcollagen with OVA challenges (FIG. 40 ), contrary to the originalfindings. In this assay, the 100% BerriQi™ Boysenberry with apple andblackcurrant treatment produced a significant increase in the quantityof collagen compared to the other groups (FIG. 40 ). This is discussedin more detail, below.

Discussion

The i.n. OVA challenge resulted in the appearance of increasedinflammatory cells within the lung. The number of eosinophils in thelung were unaffected by BerriQi™ Boysenberry with apple and blackcurranttreatment, but the number of APCs was significantly decreased byBerriQi™ Boysenberry with apple and blackcurrant treatment. Monocytestrended towards an increase by OVA challenge and trended towards adecrease with BerriQi™ Boysenberry with apple and blackcurranttreatment.

Analysis of lung tissue to quantify the changes in collagen showed thatthe quantity of total collagen following OVA challenges was unchanged,which is inconsistent with the findings from the original assay (see,e.g., Example 1 and (74)). However, Masson's trichrome stainingindicated that OVA challenges resulted in collagen infiltrating into theairways, which was counteracted by the 100% BerriQi™ Boysenberry withapple and blackcurrant treatment.

The differences in results for the hydroxyproline assays could be due tothe differences in these assays, as already noted. In addition, it ispossible that although there was no change in the quantity of totalcollagen from OVA challenge, the location of the collagen has beenaltered, and this is being addressed by the BerriQi™ Boysenberry withapple and blackcurrant treatment. It is also possible that theobservations made for this this BerriQi™ Boysenberry with apple andblackcurrant treatment, including the increase in the amount collagen,coupled with the reduction of the appearance of collagen staining withinthe airways, is a result of tissue remodelling that occurs wheninflammation is undergoing resolution.

Example 5: Dosage Calculations for Oral Treatments

The oral treatment dosages for Examples 3 and 4 were calculated inaccordance with the following information.

TABLE 1 Dose calculations for chronic challenge mouse studies Fourtreatment groups: Dose rate in mice (mg/kg Human Equivalent Sample anddescription total anthocyanins TAC) Dose* (mg/kg) 1 BerriQi ™concentrate (1) 100% 10^(†) 0.729 (Boysenberry + Apple) 2 See Example 3 50% 5 0.364 3  25% 2.5 0.182 4 BerriQi ™ concentrate (2) 100% 10 0.729(Boysenberry + Blackcurrant + Apple) See Example 4 Dosing of BerriQi ™concentrate based on 200 μL for a 25 g mouse being equivalent for a 70kg human (in total anthocyanins) ^(†)See Example 1 and (74)$\quad\begin{matrix}{*{HED}} & {= {{animal}\mspace{14mu}{dose}\mspace{14mu}{in}\mspace{14mu}{mg}\text{/}{kg} \times ( {{animal}\mspace{14mu}{weight}\mspace{14mu}{in}\mspace{14mu}{kg}\text{/}{human}} }} \\\; &  \mspace{31mu}{{weight}\mspace{14mu}{in}\mspace{14mu}{kg}} )^{0.33} \\{{For}\mspace{14mu}{example}} & {= {10\mspace{14mu}{mg}\text{/}{kg}\mspace{14mu}{mouse}\mspace{14mu}{dose} \times ( {0.025\mspace{14mu}{kg}\mspace{14mu}{mouse}\text{/}70\mspace{14mu}{kg}} }} \\\; &  \mspace{31mu}{human} )^{0.33} \\\; & {= {0.73\mspace{14mu}{mg}\text{/}{kg}\mspace{14mu}{total}\mspace{14mu}{anthocyanins}\mspace{14mu}{for}\mspace{14mu} 70\mspace{14mu}{kg}\mspace{14mu}{human}}}\end{matrix}$

TABLE 2 Starting concentrates and their composition Sample DescriptionComposition Sample BerriQi ™ 27% Boysenberry juice concentrate (1)Concentrate (1) 65 Brix (Boysenberries NZ Ltd); (Boysenberry + 72.88%Apple juice concentrate (clear) Apple) 70 Brix (RD2 International);0.12% Potassium sorbate Sample BerriQi ™ 13.5% Boysenberry juiceconcentrate (2) Concentrate (2) 65 Brix (Boysenberries NZ Ltd);(Boysenberry + 13.5% Blackcurrant juice concentrate Blackcurrant + 65Brix (NZ Blackcurrant Co-op); Apple) 72.88% Apple juice concentrate(clear) 70 Brix (RD2 International); 0.12% Potassium sorbate

TABLE 3 Calculations for anthocyanin concentrations Total anthocyanins(as cyanidin- Sample Description 3-glucoside) Calculation SampleBerriQi ™ 145 mg/100 g Specific gravity = 1.34 g/ml (1) Concentratejuice 1 g juice concentrate = 0.746 ml (1) (Boysen- concentrate 1 mljuice concentrate = 1.34 g berry + Therefore: Apple) 1,450 μg/g (1.45mg/g) or 1,943 μg/ml juice concentrate Sample BerriQi ™ 236 mg/100 gSpecific gravity = 1.34 g/ml (2) Concentrate juice 1 g juice concentrate= 0.746 ml (2) (Boysen- concentrate 1 ml juice concentrate = 1.34 gberry + Therefore: Black- 2,360 μg/g (2.36 mg/g) or currant + 3,162μg/ml juice concentrate Apple)

TABLE 4 Dosage calculations based on anthocyanin concentrations Doserate in mice Volume of BerriQi ™ (mg/kg total Concentrate required inSample Description anthocyanins TAC) 200 μL dose (μL) 1 BerriQi ™ 100%10 129 2 Concentrate (1)  50% 5 Serial dilution 3 (Boysenberry +  25%2.5 Serial dilution Apple) 4 BerriQi ™ 100% 10  79 Concentrate (2)(Boysenberry + Blackcurrant + Apple)

Detailed calculations for dosages in Table 1 for BerriQi™ concentrate(1) at 100% as utilised in Example 3 (Boysenberry with apple):

Calculation to Determine Mouse Dosage

Desire a dose of 10 mg anthocyanin/kg for a 25 g mouse (Example 1 and(74))−100%

A 10 mg anthocyanin/kg dose for a 25 g mouse would require 0.25 mg ofanthocyanin

Therefore:  0.172  g  BerriQi^()  conc (1)  required  to  deliver  a  0.25  mg  dose  of  anthocyanin = 0.129  mL  BerriQi^()  conc (1)  required  to  deliver  a  0.25  mg  dose   anthocyanin = 128.667  µL  BerriQi^()  conc (1)  required  to  deliver  a   0.25  mg  dose  anthocyanin   Sp  Gravity  1.34  g/mlPreparation of Oral Composition for Mouse TrialTake 128.667 μL BerriQi™ conc (1) and make it up to 200 μL with H₂OThere is now 0.25 mg of anthocyanin in 200 μL mouse dose, 10 mg/kg for a25 g mouse

Detailed calculations for dosages in Table 1 for BerriQi™ concentrate(2) at 100% as utilised in Example 4 (Boysenberry with apple andblackcurrant):

Calculation to Determine Mouse Dose

Desire a dose of 10 mg anthocyanin/kg for a 25 g mouse (Example 1 and(74))−100%

A 10 mg anthocyanin/kg dose for a 25 g mouse would require 0.25 mg ofanthocyanin

Therefore:  0.106  g  BerriQi^()  conc (2)  required  to  deliver  a  0.25  mg  dose  of  anthocyanin = 0.079  mL  BerriQi^()  conc (2)  required  to  deliver  a  0.25  mg  dose  of  anthocyanin = 79.054  µL  BerriQi^()  conc (2)  required  to  deliver  a   0.25  mg  dose  of  anthocyanin  Sp  Gravity  1.340  g/mlPreparation of Oral Composition for Mouse TrialTake 79.054 μL BerriQi™ cone (2) and make it up to 200 μL with H₂OThere is now 0.25 mg of anthocyanin in 200 μL mouse dose, 10 mg/kg for a25 g mouse

Alternative dosage calculation details for BerriQi™ concentrate (1)(Boysenberry+Apple):

Mouse weight 25 g

Dose (total anthocyanins) 10 mg/kg (or 0.1 mg/10 g)

Therefore: dose=0.25 mg per mouse

BerriQi™ (Boysenberry+Apple) total anthocyanins 145 mg/100 g (or 1.45mg/g)

Weight product needed 0.25 mg/1.45 mg=0.172 g BerriQi™

Able to do weight of product in water because specific gravity is known

Specific Gravity 1.34 kg/L (or 1.34 g/mL)

Volume needed 0.172 g/1.34 g=0.128.66 mL

This equates to 129 μL per mouse plus 71 μL water (200 μl minus 129 μl)

Example 6: Compositional Analysis of Treatment Formulations

BerriQi™ liquid formulations were subjected to chemical analysis todetermine anthocyanin and phenolic composition. The formulations testedincluded: Sample 1, BerriQi™ with apple concentrate 1 (BB+AP); Sample 2,BerriQi™ with apple plus blackcurrant concentrate 2 (BB+BC+AP). SeeExample 5. Weighed aliquots of the samples were diluted 5-fold with 10%formic acid_(aq) for analysis by ultra high pressure liquidchromatography (UHPLC). For analysis of other phenolics by liquidchromatography mass spectrometry (LC-MS), samples were diluted 10-foldwith 0.1% formic acid_(aq). Sample density was determined and samplestaken for dry matter calculations.

UHPLC analysis of anthocyanins: Anthocyanin concentrations were measuredusing a Dionex UltiMate 3000 Series UHPLC (ThermoFisher Scientific, SanJose, Calif., USA) with PDA (photodiode array) detection at 520 nm.Compound separation was achieved using a Synergi 4μ hydro-RP 80A column,4.6×250 mm (Phenomenex, Torrance, Calif., USA), maintained at 40° C.Solvents were (A) 5:5:90 acetonitrile:formic acid:water v/v/v and (B)5:95 v/v formic acid: acetonitrile and the flow rate was 1 mL/min. Theinitial mobile phase, 100% A was held for 1 min, then ramped linearly to84% A in 16 min, followed by a column flush at 5% A before resetting tothe original conditions. The sample injection volume was 0.5 μL.Detected anthocyanins were quantified by UHPLC using a pure standard ofcyanidin 3-O-glucoside and all the results for individual and totalanthocyanins are expressed as cyanidin 3-O-glucoside equivalents.

LC-MS confirmation: LC-MS employed an LTQ linear ion trap massspectrometer fitted with an ESI interface (ThermoFisher Scientific, SanJose, Calif., USA) coupled to an Ultimate 3000 UHPLC and PDA detector(Dionex, Sunnyvale, Calif., USA).

Anthocyanin confirmation: Anthocyanin compound separation was achievedusing a Poroshell 120 SB-C18 column, 2.7μ 2.1×150 mm (Agilent, Torrance,Calif., USA), maintained at 70° C. Solvents were (A) 5:3:92acetonitrile:formic acid:water v/v/v and (B) acetonitrile+0.1% formicacid, and the flow rate was 200 μL/min. The initial mobile phase, 100% Awas held for 2 min before being ramped linearly to 88% A at 14 min, 5% Aat 15 min and held for 4 min before resetting to the originalconditions. The sample injection volume was 10 μL. MS data were acquiredin the positive mode using a data-dependent LC-MS³ method. This methodisolates and fragments the most intense parent ion to give MS² data(daughter ions), then isolates and fragments the most intense daughterion (MS³ data).

Other phenolics: Other phenolic compound separation was achieved using aHypersil GOLD aQ 1.9μ C18 175 Å (Thermo Scientific, Waltham, Mass. USA),150×2.1 mm column maintained at 45° C. Solvents were (A) water+0.1%formic acid and (B) acetonitrile+0.1% formic acid, and the flow rate was200 μl/min. The initial mobile phase, 95% A/5% B, was ramped linearly to85% A at 10 min, held for 3.75 min, then ramped linearly to 75% A at 18min, 67.2% A at 25 min, 50% A at 28 min, 3% A at 29 min and held for 4min before resetting to the original conditions. The sample injectionvolume was 4 μL. UV-vis detection was by absorbance at 200-600 nm. MSdata were acquired in both negative and positive modes with ESIionisation using three data-dependent LC-MS³ methods, the first usingmass range [m/z 150-900] optimised for detection of low molecular weightphenolic compounds, the second using mass range [m/z 150-2000] optimisedfor detection of ellagitannins and the third using mass range [m/z150-4000] optimised for detection of higher molecular weight solubletannins. MS data were also acquired in both negative and positive modeswith APCI ionisation.

Phenolic acids, gallic acid, protocatechuric acid, chlorogenic acid(3-caffeoylquinic acid), caffeic acid were quantified by LC-MS usingpure standards of these compounds. Detected derivatives of coumaric acidwere quantified by LC-MS using p-coumaric acid, and are expressed asp-coumaric acid equivalents. The flavan-3-ols, epi-catechin andcatechin, and procyanidin B2, were quantified by LC-MS using purestandards of these compounds. Unknowns m/z 563 and m/z 579 werequantified by LC-MS using epi-catechin, and expressed as epi-catechinequivalents. Hydrolysable tannins were quantified by LC-MS using astandard of Sanguiin H6 that had been isolated previously (>98% purityby LC-MS). Other detected tannins and unknown m/z 639 were quantified byLC-MS as Sanguiin H6 equivalents. Ellagic acid was quantified by LC-MSusing a standard of ellagic acid. Detected flavonol glycosides werequantified by LC-MS using a pure standard of quercetin 3-O-glucoside andare expressed as quercetin 3-O-glucoside equivalents. Thenon-glycosylated flavanols, quercetin and myricetin, and the chalcones,phloretin and phloretin-2-O-glucoside were quantified by LC-MS usingpure standards of these compounds. The preservative sorbic acid wasquantified by LC-MS using a pure standard of this compound.

The results for the analysis are shown as follows.

TABLE 5 Density and dry matter data for BerriQi ™ samples Density DryMatter Sample Description (g/mL) (%) 1 BerriQi ™ concentrate 1.346 70.24(1) BB + AP 2 BerriQi ™ concentrate 1.351 71.37 (2) BB + BC + AP

TABLE 6 Quantitation summary for detected phenolics in BerriQi ™ samples1 and 2 expressed in μg/mL and μg/g dry weight (DW) Sample 1 Sample 2μg/g μg/g Compound μg/mL DW μg/mL DW Anthocyanins Delphinidin3-O-glucoside nd nd 120 125 Cyanidin 3-O-sophoroside 925 978 306 317Delphinidin 3-O-rutinoside nd nd 868 900 Cyanidin 3-O-glucoside 597 631248 258 Cyanidin 3-O-sambubioside 26 28 8 8 Cyanidin3-O-(2-glucosylrutinoside) 431 456 146 152 Cyanidin 3-O-rutinoside 69 73913 947 Cyanidin 3-O-xylosylrutinoside 18 19 5 5 Phenolic acids Gallicacid 162 171 33 35 Protocatechuric acid 41 43 17 18 Chlorogenic acid 7478 60 63 Caffeic acid 6 6 6 6 4-p-Coumaroylquinic acid 20 21 33 355-p-Coumaroylquinic acid 3 3 8 9 Flavan-3-ols and procyanidinsProcyanidin B2 5 5 6 7 Catechin 3 3 5 5 Epi-catechin 17 18 11 12Hydrolysable tannins Sanguiin H10 isomer 1 13 13 6 7 Sangui sorbic aciddilactone 234 248 51 52 Galloyl-SH6 79 84 24 25 Sanguiin H10 isomer 2 7984 44 45 Lambertian C (minus ellagic acid) 13 14 9 9 Lambertian C 27 299 10 Sanguiin H6 257 271 99 103 Ellagic acid 626 662 96 100 FlavanolsQuercetin 3-O-rutinoside 8 8 62 64 Quercetin 3-O-galactoside 18 19 26 27Quercetin 3-O-glucuronide 42 45 3 3 Quercetin 3-O-glucoside 13 14 36 37Quercetin 3-O-pentoside 1 9 10 12 12 Quercetin 3-O-pentoside 2 11 12 1415 Quercetin 3-O-pentoside 3 6 7 3 4 Quercetin 3-O-rhamnoside 14 13 1513 Quercetin 33 35 37 38 Myricetin-3-O-rutinoside nd nd 81 84Myricetin-3-O-glucoside nd nd 33 35 Myricetin-malonylglucoside nd nd 4 4Myricetin nd nd 7 7 Aureusidin-glucoside nd nd 3 3Kaempferol-3-O-rutinoside nd nd 8 8 Kaempferol-3-O-glucoside nd nd 5 5Chalcones Phloretin 2-O-xylo-glucoside 10 11 9 10 Phloretin2-O-glucoside 76 80 95 99 Unknowns Unknown m/z 563# 10 11 4 4 Unknownm/z 639 65 69 54 56 Unknown m/z 579 3 3 3 3 Totals 4043 4275 3645 3784nd = not detected # = detected as [M + formate] − adduct

Persons of ordinary skill can utilise the disclosures and teachingsherein to produce other embodiments and variations without undueexperimentation. All such embodiments and variations are considered tobe part of this invention.

Accordingly, one of ordinary skill in the art will readily appreciatefrom the disclosure that later modifications, substitutions, and/orvariations performing substantially the same function or achievingsubstantially the same result as embodiments described herein may beutilised according to such related embodiments of the present invention.Thus, the invention is intended to encompass, within its scope, themodifications, substitutions, and variations to processes, manufactures,compositions of matter, compounds, means, methods, and/or stepsdisclosed herein.

The description herein may contain subject matter that falls outside ofthe scope of the claimed invention. This subject matter is included toaid understanding of the invention.

In this specification, where reference has been made to external sourcesof information, including patent specifications and other documents,this is generally for the purpose of providing a context for discussingthe features of the present invention. Unless stated otherwise,reference to such sources of information is not to be construed, in anyjurisdiction, as an admission that such sources of information are priorart or form part of the common general knowledge in the art.

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What is claimed is:
 1. A method for: (i) treating an airway inflammationin a human in need thereof; (ii) treating accumulation of collagenfibres in lung tissue of a human in need thereof; or (iii) treatingimmune cell infiltration in lung tissue of a human in need thereof; themethod comprising administering to the human a nutraceutical compositioncomprising: (a) a combined Boysenberry juice concentrate and apple juiceconcentrate, wherein the percentage of the Boysenberry juice concentrateto the apple juice concentrate is about 17% to about 34% v/v ofBoysenberry juice concentrate to about 83% to about 66% v/v of applejuice concentrate; or (b) a combined Boysenberry juice concentrate,apple juice concentrate, and blackcurrant juice concentrate, wherein thepercentage of the Boysenberry and blackcurrant juice concentrate to theapple juice concentrate is about 17% to about 34% v/v of Boysenberry andblackcurrant juice concentrate to about 83% to about 66% v/v of applejuice concentrate, thereby effectively treating the airway inflammation,the accumulation of collagen fibres in the lung tissue, or the immunecell infiltration in the lung tissue.
 2. The method of claim 1, whereinthe nutraceutical composition is administered to the human once per day.3. The method of claim 1, wherein the nutraceutical compositioncomprises a dosage unit comprising about 5 mg to about 500 mg totalanthocyanins.
 4. The method of claim 1, wherein the nutraceuticalcomposition is administered enterally or orally.
 5. The method of claim1, wherein the nutraceutical composition is administered as a syrup,drop, gel, jelly, tablet, or capsule.
 6. The method of claim 1, whereinthe nutraceutical composition is administered at: (i) a dosage of about0.1 mg/kg to about 10 mg/kg total anthocyanins per human's body weight;or (ii) a dosage of about 0.1 mg/kg to about 5 mg/kg total anthocyaninsper human's body weight; or (iii) a dosage of about 10 mg to about 200mg total anthocyanins per day.
 7. The method of claim 1, wherein thenutraceutical composition further comprises added polyphenols.
 8. Themethod of claim 1, wherein the nutraceutical composition is administeredwith a respiratory aid.
 9. The method of claim 1, wherein the human hasa chronic respiratory disorder.
 10. The method of claim 1, wherein thehuman has allergic airways inflammation or reactive airway disease. 11.The method of claim 1, wherein the immune cell is selected from thegroup consisting of monocytes, eosinophils, neutrophils, and antigenpresenting cells.
 12. The method of claim 1, wherein the nutraceuticalcomposition is administered to the human at a dosage of (i) about 10 mgto about 200 mg total Boysenberry anthocyanins per day, or (ii) about 10mg to about 200 mg total Boysenberry and blackcurrant anthocyanins perday.
 13. The method of claim 1, wherein a juice concentrate volume inthe nutraceutical composition is adjusted to include less than 1% v/vpreservative.
 14. The method of claim 8, wherein the further respiratoryaid is a medication, a herbal remedy, or an essential oil.
 15. Themethod of claim 8, wherein the further respiratory aid has one or moreof anti-inflammatory, anti-spasmodic, bronchodilation, or musclerelaxation effects.